Rescue of mumps virus from cDNA

ABSTRACT

This invention relates to a method for recombinantly producing, via rescue of mumps virus, a nonsegmented, negative-sense, single-stranded RNA virus, and immunogenic compositions formed therefrom. Additional embodiments relate to methods of producing the mumps virus as an attenuated and/or infectious virus. The recombinant viruses are prepared from cDNA clones, and, accordingly, viruses having defined changes, including nucleotide/polynucleotide deletions, insertions, substitutions and re-arrangements, in the place of the genome are obtained.

This application is a National Stage entry of PCT/US00/21192 filed Aug.2, 2000.

FIELD OF THE INVENTION

This invention relates to a method for recombinantly producing mumpsvirus, a nonsegmented, negative-sense, single-stranded RNA virus, andimmunogenic compositions formed therefrom. Additional embodiments relateto methods of producing the mumps virus as an attenuated and/orinfectious virus. The recombinant viruses are prepared from cDNA clones,and, accordingly, viruses having defined changes in the genome areobtained. This invention also relates to use of the recombinant virusformed therefrom as vectors for expressing foreign genetic information,e.g. foreign genes, for many applications, including immunogenic orpharmaceutical compositions for pathogens other than mumps, genetherapy, and cell targeting.

BACKGROUND OF THE INVENTION

Enveloped, negative-sense, single stranded RNA viruses are uniquelyorganized and expressed. The genomic RNA of negative-sense, singlestranded viruses serves two template functions in the context of anucleocapsid: as a template for the synthesis of messenger RNAs (mRNAs)and as a template for the synthesis of the antigenome (+) strand.Negative-sense, single stranded RNA viruses encode and package their ownRNA-dependent RNA Polymerase. Messenger RNAs are only synthesized oncethe virus has entered the cytoplasm of the infected cell. Viralreplication occurs after synthesis of the mRNAs and requires thecontinuous synthesis of viral proteins. The newly synthesized antigenome(+) strand serves as the template for generating further copies of the(−) strand genomic RNA.

The etiological agent of mumps was first shown reproducibly to be avirus by Johnson and Goodpasture in 1935 (Johnson and Goodpasture,1935). Since then, propagation in tissue culture has facilitated virusclassification and studies on the biological properties of mumps virus(MUV). Originally classified with influenza viruses in the Myxovirusfamily, mumps virus has since been re-assigned to the Paramyxoviridaefamily, subfamily Paramyxovirinae, genus Rubulavirus, based onnucleocapsid morphology, genome organization and biological propertiesof the proteins. Other examples of the Rubulavirus genus include simianvirus 5 (SV5), human parainfluenza virus type 2 and type 4 and Newcastledisease virus (Lamb and Kolakofsky, 1996). Like all viruses of theParamyxoviridae, mumps virus is pleomorphic in shape, comprising a hostcell derived lipid membrane surrounding a ribonucleoprotein core; thisnucleocapsid core forms a helical structure composed of a 15,384nucleotide nonsegmented negative sense RNA genome closely associatedwith virus nucleocapsid protein (NP). The genetic organization of theMUV genome has been determined to be 3′-NP-P-M-F-SH-HN-L-5′ (Elango etal., 1998). Each gene encodes a single protein except for the P cistron,from which three unique mRNAs are transcribed; one is a faithful copy ofthe P gene, encoding the V protein, the two other mRNAs contain two andfour non-templated G residues inserted during transcription by a RNAediting mechanism, and encode the P and I proteins respectively(Paterson and Lamb, 1990). The P and L proteins in association withnucleocapsid form the functional RNA polymerase complex of mumps virus.The F and HN proteins are integral membrane proteins which project fromthe surface of the virion, and are involved in virus attachment andentry of cells. The small hydrophobic protein (SH) and matrix (M)protein are also membrane associated (Takeuchi et al, 1996 and Lamb andKolakofsky, 1996); the role of the V and I proteins in virus growth isnot yet clear.

The replicative cycle of mumps virus initiates upon fusion of virusenvelope with host cell plasma membrane and subsequent release of virusnucleocapsid into the cell cytoplasm. Primary transcription then ensues,resulting in the production of all virus proteins; a switch toreplication of the virus genome occurs later, followed by assembly ofvirus components to form new virus particles which bud from the hostcell plasma membrane. Only the intact nucleocapsid structure can act asthe template for RNA transcription, replication and subsequent virusamplification; therein lies the difficulty in genetic manipulation ofMUV and other negative strand RNA viruses. Unlike the positive strandRNA viruses where naked genomic RNA is infectious and infectious viruscan be recovered from a cDNA copy of the genome in the absence ofadditional viral factors (Taniguchi et al., 1978; Racaniello andBaltimore, 1981), the naked genome of negative strand RNA viruses is notinfectious and rescue of virus from cDNA requires intracellularco-expression of viral NP, P and L proteins, along with a full lengthpositive sense, or negative sense, genome RNA transcript, all undercontrol of the bacteriophage T7 RNA polymerase promoter (Schnell et al.,1994; Lawson et al. 1995; Whelan et al., 1995; Radecke et al., 1995;Collins et al., 1995; Hoffman and Banerjee, 1997; Durbin et al., 1997;He et al., 1997; Baron and Barrett, 1997; Jin et al., 1998; Buchholz etal., 1999; Peeters et al., 1999). In all of the reported systems T7 RNApolymerase has been supplied either by a co-infecting recombinantvaccinia virus (Fuerst et al., 1986; Wyatt et al., 1995), or byendogenous expression of T7 RNA polymerase in a transformed cell line(Radecke et al., 1995).

The polymerase complex actuates and achieves transcription andreplication by engaging the cis-acting signals at the 3′ end of thegenome, in particular, the promoter region. Viral genes are thentranscribed from the genome template unidirectionally from its 3′ to its5′ end. There is generally less mRNA made from the downstream genes(e.g., the polymerase gene (L)) relative to their upstream neighbors(i.e., the nucleoprotein gene (NP)). Therefore, there is always agradient of mRNA abundance according to the position of the genesrelative to the 3′-end of the genome.

Molecular genetic analysis of such nonsegmented RNA viruses has proveddifficult until recently because naked genomic RNA or RNA producedintracellularly from a transfected plasmid is not infectious (Boyer andHaenni, 1994). These methods are referred to herein as “rescue”. Thereare publications on methods of manipulating cDNA rescue methods thatpermit isolation of some recombinant nonsegmented, negative-strand RNAviruses (Schnell et al., 1994). The techniques for rescue of thesedifferent negative-strand viruses follows a common theme; however, eachvirus has distinguishing requisite components for successful rescue(Baron and Barrett, 1997; Collins et al., 1995; Garcin et al., 1995;Hoffman and Banerjee, 1997; Lawson et al., 1995; Radecke et al., 1995;Schneider et al., 1997; He et al, 1997; Schnell et al., 1994; Whelan etal., 1995). After transfection of a genomic cDNA plasmid, an exact copyof genome RNA is produced by the combined action of phage T7 RNApolymerase and a vector-encoded ribozyme sequence that cleaves the RNAto form the 3′ termini. This RNA is packaged and replicated by viralproteins initially supplied by co-transfected expression plasmids. Inthe case of the mumps virus, a method of rescue has yet to beestablished and accordingly, there is a need to devise a method of mumpsrescue. Devising a method of rescue for mumps virus is complicated bythe absence of extensive studies on the biology of mumps virus, ascompared with studies on other RNA viruses. Also, mumps virus does notgrow efficiently in tissue culture systems. Furthermore, the sequencefor the termini of the mumps virus genome has not previously beencharacterized in sufficient detail for conducting rescue.

For successful rescue of mumps virus from cDNA to be achieved, numerousmolecular events must occur after transfection, including: 1) accurate,full-length synthesis of genome or antigenome RNA by T7 RNA polymeraseand 3′ end processing by the ribozyme sequence; 2) synthesis of viralNP, P, and L proteins at levels appropriate to initiate replication; 3)the de novo packaging of genomic RNA into transcriptionally-active andreplication-competent nucleocapsid structures; and 4) expression ofviral genes from newly-formed nucleocapsids at levels sufficient forreplication to progress.

The present invention provides for a rescue method of recombinantlyproducing mumps virus. The rescued mumps virus possesses numerous uses,such as antibody generation, diagnostic, prophylactic and therapeuticapplications, cell targeting, mutant virus preparation and immunogeniccomposition preparation. Furthermore, there are a number of advantagesto using a recombinantly produced Jeryl Lynn strain of mumps for theseapplications. Some of these advantages include (1) an attenuatedphenotype, (2) a substantial safety record based on the over 100 milliondosages administered, (3) the ability to induce long-lasting immunitywith a single dose and (4) a relatively low level of genomerecombination.

SUMMARY OF THE INVENTION

The present invention provides for a method for producing a recombinantmumps virus comprising, in at least one host cell, conductingtransfection of a rescue composition which comprises (i) a transcriptionvector comprising an isolated nucleic acid molecule which comprises apolynucleotide sequence encoding a genome or antigenome of a mumps virusand (ii) at least one expression vector which comprises at least oneisolated nucleic acid molecule encoding the trans-acting proteinsnecessary for encapsidation, transcription and replication. Thetransfection is conducted under conditions sufficient to permit theco-expression of these vectors and the production of the recombinantvirus. The recombinant virus is then harvested.

Additional embodiments relate to the nucleotide sequences, which uponmRNA transcription express one or more, or any combination of, thefollowing proteins of the mumps virus: NP, M, F, SH, HN L and the V, P,and I proteins which are generated from the P “cistron” of mumps virusas noted above. Related embodiments relate to nucleic acid moleculeswhich comprise such nucleotide sequences. A preferred embodiment of thisinvention are the nucleotide sequences of SEQ ID NOS. 1, 11 and 12.Further embodiments relate to these nucleotides, the amino acidssequences of the above mumps virus proteins and variants thereof.

The protein and nucleotide sequences of this invention possessdiagnostic, prophylactic and therapeutic utility for mumps virus. Thesesequences can be used to design screening systems for compounds thatinterfere or disrupt normal virus development, via encapsidation,replication, or amplification. The nucleotide sequence can also be usedin the preparation of immunogenic compositions for mumps virus and/orfor other pathogens when used to express foreign genes. In addition, theforeign genes expressed may have therapeutic application.

In preferred embodiments, infectious recombinant virus is produced foruse in immunogenic compositions and methods of treating or preventinginfection by mumps virus and/or infection by other pathogens, whereinthe method employs such compositions.

In alternative embodiments, this invention provides a method forgenerating recombinant mumps virus which is attenuated, infectious orboth. The recombinant viruses are prepared from cDNA clones, and,accordingly, viruses having defined changes in the genome can beobtained. Further embodiments employ the consensus genome sequenceand/or any of the genome sequences within the population of the JerylLynn strain of mumps to express foreign genes since this licensedvaccine strain includes an established attenuated phenotype for safety.Since the consensus sequence is derived from a proposed average of thegenomes of mumps virus, the polynucleotide sequences for the genomeswithin the population of the Jeryl Lynn strain are embodiments of thisinvention.

This invention also relates to use of the recombinant virus formedtherefrom as vectors for expressing foreign genetic information, e.g.foreign genes, for many applications, including immunogenic compositionsfor pathogens other than mumps, gene therapy, and cell targeting.

The above-identified embodiments and additional embodiments, which arediscussed in detail herein, represent the objects of this invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a diagram showing the organization of the MUVCATminireplicon DNA construct and T7 RNA polymerase-transcribedminireplicon antisense RNA genome. Key restriction endonuclease sitesutilized in the assembly of the DNA construct are shown. The T7 RNApolymerase promoter sequence was designed to start transcription withthe exact MUV 5′ terminal nucleotide, and a HDV ribozyme sequence waspositioned to generate the precise MUV 3′ terminal nucleotide inminireplicon RNA transcripts. Duplicate T7 RNA polymerase terminationsignals were included in tandem after the HDV ribozyme sequence. The CATORF replaces all of the coding and intercistronic sequence of the MUVgenome; the remaining essential MUV specific sequence comprises the 3′MUV Leader (55 nt) with adjacent 90 nt NP gene untranslated region(UTR), and the 5′ MUV Trailer (24 nt) adjacent to the 137 nt L gene UTR.

FIG. 2 is a schematic representation of the MUV full-length genome cDNAconstruct, including the sub-genomic fragments and restrictionendonuclease sites used in the assembly process. The T7 RNA polymerasepromoter and the HDV ribozyme sequence were positioned to initiatetranscription with the exact 5′ terminal nucleotide and generate theprecise 3′ terminal nucleotide of the MUV antisense genome,respectively. Tandem T7 RNA polymerase termination sequences were placedadjacent to the HDV ribozyme to help improve the efficiency of RNAcleavage. Nucleotide substitutions utilized as identifying tags forrescued MUV are shown at Table 1 (See FIG. 8).

FIG. 3A depicts three thin layer chromatograms that show CAT activitypresent in 293 cells following infection with MUV and transfection withRNA transcribed in vitro from pMUVCAT as described in Example 2.

FIG. 3B depicts thin layer chromatograms showing CAT activity in MVA-T7infected Hep2 and A549 cells following transfection with pMUVCAT andplasmids expressing MUV NP, P and L proteins. The level of pMUVNPexpression plasmid was titrated in both cell lines; lanes 1-4 show CATactivity following transfection with mixtures containing 200 ng pMUVCAT,50 ng pMUVP, 200 ng pMUVL each, and 300 ng, 450 ng, 600 ng, 750 ngpMUVNP respectively; lane 5 shows CAT activity produced when pMUVL wasomitted from the transfection mixture.

FIG. 4 depicts the Passage (P1) of transfected cell supernatants on A549cells, as described in Example 3. Views A, B and C correspond to rescuedmumps virus, no mumps virus (control) and Jeryl Lynn strain of mumps.The views show similar infectious foci for A and C.

FIG. 5 depicts a whole cell ELISA of rescued mumps virus on a Vero cellmonolayer, as described in Example 3.

FIG. 6 shows the gel analysis of RT/PCR products used to identify rMUV(as described in Example 4). Total RNA was prepared from Vero cellmonolayers infected with passage 2 of rMUV virus from transfected cells.RT/PCR reactions were set up to generate cDNA products spanning the 3separate nucleotide tag sites present only in pMUVFL and rMUV. Lane 1shows marker 1 kb ladder (Gibco/BRL); lanes 2, 3 and 4 show RT/PCRproducts spanning nucleotide tag positions 6081, 8502 and 11731,respectively. To demonstrate that these RT/PCR products were not derivedfrom contaminating plasmid DNAs, an identical reaction to that used forthe generation of the cDNA shown in lane 4 was performed without RT; theproduct(s) of this reaction are shown in lane 5. To demonstrate that norMUV could be recovered when pMUVL was omitted from transfectionmixtures, a RT/PCR reaction identical to that used to generate the cDNAproducts shown in lane 4 was set up using Vero cell RNA derived fromtransfections carried out without pMUVL; products from this reaction areshown in lane 6.

FIG. 7 depicts three electropherograms (A, B, and C) showing nucleotidesequence across identifying tag sites in rMUV. RT/PCR products (FIG. 6),which were sequenced across each of the three tag sites. The nucleotidesequence at each tag site obtained for rMUV cDNA is compared withconsensus sequence for the plaque isolate of MUV (plaque isolate 4, PI4) used to derive pMUVFL.

FIG. 8 is a table (Table 1) that lists the nucleotide and amino aciddifferences between the full length cDNA clone and the plaque isolate 4(PI4) and the consensus sequence for the Jeryl Lynn strain (SEQ ID NO.1).

FIG. 9 is a table (Table 2) which describes a complete gene map formumps virus, including the gene start and gene end for mumps virusproteins. The sequence of the 55 nucleotide long 3′ leader and 24nucleotide long 5′ trailer are also shown.

FIG. 10 is a table (Table 3) that lists the mumps virus genetranscription start and stop nucleotide positions, along with thetranslation start and stop positions for the individual genes of themumps genome as provided in SEQ ID NO 1. The nucleotides from eachtranscription (gene) start and to each stop nucleotide position in Table3 correspond to nucleotide sequences for proteins NP, P, M, F, SH, HNand L (SEQ ID NOS 93-99, respectively).

FIG. 11 is a diagram showing the insertion of the luciferase andbeta-galactosidase gene(s) into the mumps virus genome between the M andthe F genes. An AscI site was generated by site directed mutagenesis inthe 5′ non-coding region of the M gene. Nested PCR was used to generatemumps virus specific M-F intergenic sequence(s) and terminal AscI sitesflanking each reporter gene. The resulting PCR product(s) were digestedwith AscI and imported into the genome AscI site.

FIG. 12 is a diagram showing the insertion of two genes (luciferase andCAT) into the mumps virus genome. Two separate transcription units and asingle transcription unit containing an internal ribosomal entry sitefor expression of the second gene of the polycistron, were separatelyinserted into the AscI site present in the M-F intergenic region. NestedPCR was used to generate the appropriate mumps virus M-F intergenicsequence flanking each gene and transcriptional unit.

FIG. 13 depicts the results from the MAPREC analysis of ten Mumpsvax®vaccine samples for relative portions of JL5/JL2 as determined from RNAwas isolated from ten vials of mumps Jeryl Lynn vaccine and amplified byRT-PCR, as described in Example 7. The tested samples in Lanes 1 and 2are serial dilutions of undigested PCR product used to define the lowerlimits of linearity for the assay. In Lane 3 the PCR product is from apurified isolate of JL5. In Lane 4, the PCR product is from a purifiedisolate of JL2. In Lanes 5-8, the PCR products are from samples of JL5and JL2 viruses mixed in the following ratios: 99 JL5/1 JL2, 95 JL5/5JL2, 85 JL5/15 JL2, and 75 JL5/25 JL2, respectively. For Lanes 9-18, thePCR products are from Mumpsvax® samples 1-10.

FIG. 14 depicts a thin layer chromatogram that shows CAT activitypresent in the extracts of Vero cells which were infected with rMUVcontaining both the CAT and luciferase genes, as described in Example 5.

FIG. 15 is a photograph showing cytological staining of Vero cellmonolayers which were infected with rMUV containing thebeta-galactosidase gene, as described n Example 5. The presence ofintense blue stain indicated beta-galactosidase expression and activity.Panel C also shows a “clear” plaque made by rMUV which did not containany additional foreign genes.

BRIEF SUMMARY OF PRIMARY SEQUENCES

Sequence 1 is the consensus nucleotide sequence for the full-lengthgenome for Jeryl Lynn strain of mumps virus. (SEQ ID NO. 1), which iswritten in the antigenomic (+, 5′ to 3′), message sense.

Sequence 2 is the amino acid sequence of the mumps virus Jeryl Lynnstrain NP protein. (SEQ ID NO. 2)

Sequence 3 is the amino acid sequence of the mumps virus Jeryl Lynnstrain P protein. (SEQ ID NO 3)

Sequence 4 is the amino acid sequence of the mumps virus Jeryl Lynnstrain I protein. (SEQ ID NO 4)

Sequence 5 is the amino acid sequence of the mumps virus Jeryl Lynnstrain V protein. (SEQ ID NO 5)

Sequence 6 is the amino acid sequence of the mumps virus Jeryl Lynnstrain M protein. (SEQ ID NO 6)

Sequence 7 is the amino acid sequence of the mumps virus Jeryl Lynnstrain F protein. (SEQ ID NO 7)

Sequence 8 is the amino acid sequence of the mumps virus Jeryl Lynnstrain SH protein. (SEQ ID NO 8)

Sequence 9 is the amino acid sequence of the mumps virus Jeryl Lynnstrain HN protein. (SEQ ID NO 9)

Sequence 10 is the amino acid sequence of the mumps virus Jeryl Lynnstrain L protein. (SEQ ID NO 10)

Sequence 11 is the complete nucleotide sequence of mumps Jeryl Lynn JL5variant for plaque 2 (SEQ ID NO 11). Plaque 1 differed from plaque 2 atposition 1703 (See Table 6). Sequence is written as DNA in antigenomic(+, 5′ to 3′) sense.

Sequence 12 is the complete nucleotide sequence of mumps Jeryl Lynn JL2variant for plaque 2 (SEQ ID NO 12). Plaque 1 differs from plaque 2 at 5nucleotide positions (See Table 7). Sequence is written as DNA inantigenomic (+, 5′ to 3′) sense.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention relates to a method of producingrecombinant mumps virus (MUV). Such methods in the art are referred toas “rescue” or reverse genetics methods. Several rescue methods fordifferent nonsegmented, negative-strand viruses are disclosed in thefollowing referenced publications: Baron and Barrett, 1997; Collins etal., 1995; Garcin et al., 1995; He et al., 1997; Hoffman and Banerjee,1997; Lawson et al., 1995; Radecke and Billeter, 1997; Radecke et al.,1995; Schneider et al., 1997; Schnell, 1994; Whelan et al., 1995.Additional publications on rescue include published International patentapplication WO 97/06270 for MV and other viruses of the subfamilyParamyxovirinae, and for RSV rescue, published International patentapplication WO 97/12032.

Before conducting rescue of recombinant mumps virus, it was necessary todevelop a consensus sequence for the entire mumps virus (Jeryl Lynnstrain) and also develop a minireplicon rescue system for mumps virus(MUV). The consensus sequence is obtained by sampling the population ofRNA genomes present during a mumps virus infection of a cell.Correspondingly, further embodiments of this invention relate to anisolated polynucleotide sequence encoding the genome or antigenome ofmumps virus or proteins thereof, as well as variants of such sequences.Preferably, under high stringency conditions, these variant sequenceshybridize to polynucleotides encoding one or more mumps proteins (SeeTable 2 of FIG. 9 for a complete map of the mumps virus, including thegene start and gene stop end for mumps virus proteins). More preferably,under high stringency conditions, these variant sequences hybridize topolynucleotides encoding one or more mumps virus strains, such as thepolynucleotide sequences of SEQ ID NOS. 1, 11 and 12. For the purposesof defining high stringency southern hybridization conditions, referencecan conveniently be made to Sambrook et al. (1989) at pp. 387-389 whichis herein incorporated by reference, where the washing step at paragraph11 is considered high stringency. This invention also relates toconservative variants wherein the polynucleotide sequence differs from areference sequence through a change to the third nucleotide of anucleotide triplet. Preferably these conservative variants function asbiological equivalents to the mumps virus reference polynucleotidereference sequence. The “isolated” sequences of the present inventionare non-naturally occurring sequences. For example, these sequences canbe isolated from their normal state within the genome of the virus; orthe sequences may be synthetic, i.e. generated via recombinanttechniques, such as well-known recombinant expression systems, orgenerated by a machine.

This invention also relates to nucleic acid molecules comprising one ormore of such polynucleotides. As noted above, a given nucleotideconsensus sequence may contain one or more of the genomes within thepopulation of a mumps virus, such as the Jeryl Lynn strain. Specificembodiments employ the consensus nucleotide sequence of SEQ ID. NOS 1,11 or 12, or nucleotide sequences, which when transcribed, express oneor more of the mumps virus proteins (NP, P/I/V, M, F, SH, HN and L). SeeTable 3 of FIG. 10 for the gene start, translation start, translationend, and gene end for these mumps virus proteins.

Further embodiments relate to the amino acid sequences for the mumpsvirus proteins NP, P/I/V, M, F, SH, HN and L as set forth in SEQ ID NOS.2-10, respectively and also to fragments or variants thereof.Preferably, the fragments and variant amino acid sequences and variantnucleotide sequences expressing mumps virus proteins are biologicalequivalents, i.e. they retain substantially the same function of theproteins in order to obtain the desired recombinant mumps virus, whetherattenuated, infectious or both. Such variant amino acid sequences areencoded by polynucleotides sequences of this invention. Such variantamino acid sequences may have about 70% to about 80%, and preferablyabout 90%, overall similarity to the amino acid sequences of the mumpsvirus protein. The variant nucleotide sequences may have either about70% to about 80%, and preferably about 90%, overall similarity to thenucleotide sequences which, when transcribed, encode the amino acidsequences of the mumps virus proteins or a variant amino acid sequenceof the mumps virus proteins. Exemplary nucleotide sequences for mumpsvirus proteins NP, P/I/V, M F, SH, HN and L are described in Tables 1and 2 (of FIGS. 8 and 9, respectively).

The biological equivalents can be obtained by generating variants of thenucleotide sequence or the protein sequence. The variants can be aninsertion, substitution, deletion or rearrangement of the templatesequence. Variants of a mumps polynucleotide sequence can be generatedby conventional methods, such as PCR mutagenesis, amino acid (alanine)screening, and site specific mutagenesis. The phenotype of the variantcan be assessed by conducting a rescue with the variant to assesswhether the desired recombinant mumps virus is obtained or the desiredbiological effect is obtained. The variants can also be assessed forantigenicity if the desired use is in an immunogenic composition.

Amino acid changes may be obtained by changing the codons of thenucleotide sequences. It is known that such changes can be obtainedbased on substituting certain amino acids for other amino acids in theamino acid sequence. For example, through substitution of alternativeamino acids, small conformational changes may be conferred upon proteinthat may result in a reduced ability to bind or interact with otherproteins of the mumps virus. Additional changes may alter the level ofattenuation of the recombinant mumps virus.

One can use the hydropathic index of amino acids in conferringinteractive biological function on a polypeptide, as discussed by Kyteand Doolittle (1982), wherein it was found that certain amino acids maybe substituted for other amino acids having similar hydropathic indicesand still retain a similar biological activity. Alternatively,substitution of like amino acids may be made on the basis ofhydrophilicity, particularly where the biological function desired inthe polypeptide to be generated is intended for use in immunologicalembodiments. See, for example, U.S. Pat. No. 4,554,101 (which is herebyincorporated herein by reference), which states that the greatest localaverage hydrophilicity of a “protein,” as governed by the hydrophilicityof its adjacent amino acids, correlates with its immunogenicity.Accordingly, it is noted that substitutions can be made based on thehydrophilicity assigned to each amino acid.

In using either the hydrophilicity index or hydropathic index, whichassigns values to each amino acid, it is preferred to introducesubstitutions of amino acids where these values are ±2, with ±1 beingparticularly preferred, and those within ±0.5 being the most preferredsubstitutions.

Preferable characteristics of the mumps virus proteins, encoded by thenucleotide sequences of this invention, include one or more of thefollowing: (a) being a membrane protein or being a protein directlyassociated with a membrane; (b) capable of being separated as a proteinusing an SDS acrylamide (10%) gel; and (c) retaining its biologicalfunction in contributing to the rescue and production of the desiredrecombinant mumps virus in the presence of other appropriate mumps virusproteins.

With the above nucleotide and amino acid sequences in hand, one can thenproceed in rescuing mumps virus. Mumps rescue is achieved by conductingtransfection, or transformation, of at least one host cell, in media,using a rescue composition. The rescue composition comprises (i) atranscription vector comprising an isolated nucleic acid molecule whichcomprises at least one polynucleotide sequence encoding a genome orantigenome of mumps virus and (ii) at least one expression vector whichcomprises one or more isolated nucleic acid molecule(s) encoding thetrans-acting proteins necessary for encapsidation, transcription andreplication; under conditions sufficient to permit the co-expression ofsaid vectors and the production of the recombinant virus. By antigenomeis meant an isolated positive message sense polynucleotide sequencewhich serves as the template for synthesis of progeny genome.Preferably, a polynucleotide sequence is a cDNA which is constructed toprovide upon transcription a positive sense version of the mumps genomecorresponding to the replicative intermediate RNA, or antigenome, inorder to minimize the possibility of hybridizing with positive sensetranscripts of complementing sequences encoding proteins necessary togenerate a transcribing, replicating nucleocapsid. The transcriptionvector comprises an operably linked transcriptional unit comprising anassembly of a genetic element or elements having a regulatory role inthe mumps expression, for example, a promoter, a structural gene orcoding sequence which is transcribed into mumps RNA, and appropriatetranscription initiation and termination sequences.

The transcription vector is co-expressed with mumps virus proteins, NP,P and L, which are necessary to produce nucleocapsid capable of RNAreplication, and also render progeny nucleocapsids competent for bothRNA replication and transcription. The NP, P and L proteins aregenerated from one or more expression vectors (e.g. plasmids) encodingthe required proteins, although one, or one or more, of these requiredproteins may be produced within the selected host cell engineered tocontain and express these virus-specific genes and gene products asstable transformants. In a preferred embodiment, NP, P and L proteinsare expressed from an expression vector. More preferably, NP, P and Lproteins are each expressed from separate expression vectors, such asplasmids. In the latter instance, one can more easily control therelative amount of each protein that is provided during transfection, ortransformation. Additional mumps virus proteins may be expressed fromthe plasmids that express for NP, P or L, or the additional proteins canbe expressed by using additional plasmids.

Although the amount of NP, P and L will vary depending on the toleranceof the host cell for their expression, the plasmids expressing NP, P andL are adjusted to achieve an effective molar ratio of NP, P and L,within the cell. The effective molar ratio is a ratio of NP, P and Lthat is sufficient to provide for successful rescue of the desiredrecombinant mumps virus. These ratios can be obtained based on theratios of the expression plasmids as observed in minireplicon(CAT/reporter) assays. In one embodiment, the molecular ratio oftransfecting plasmids pMUVNP: pMUVP is at least about 16:1 andpMUVP:pMUVL is at least about about 1:6. Preferably, the molecular ratioof pMUVNP: pMUVP is about 16:1 to about 4:1 and pMUVP:pMUVL is about 1:6to about 1:1. More preferably, the ratio of pMUVNP: pMUVP is about 6:1to about 5:1 and pMUVP:pMUVL is about 1:3 to about 1:2.

After transfection, or transformation, of a genomic cDNA plasmid alongwith mumps virus expression plasmids pMUVNP, pMUVP and pMUVL, an exactcopy of genome RNA is produced by the combined action of phage T7 RNApolymerase and a vector-encoded ribozyme sequence that cleaves the RNAto form the 3′ termini. This RNA is packaged and replicated by viralproteins initially supplied by co-transfected expression plasmids. Inthe case of the mumps virus rescue, a source that expresses T7 RNApolymerase is added to the host cell (or cell line), along with thesource(s) for NP, P and L. Mumps rescue is achieved by co-transfectingthis cell line with a mumps virus genomic cDNA clone containing anappropriately positioned T7 RNA polymerase promoter and expressionplasmid(s) that encodes the mumps virus proteins NP, P and L.

For rescue of mumps, a cloned DNA equivalent of the desired viral genomeis placed between a suitable DNA-dependent RNA polymerase promoter(e.g., the T7 RNA polymerase promoter) and a self-cleaving ribozymesequence (e.g., the hepatitis delta ribozyme) which is inserted into asuitable transcription vector (e.g a bacterial plasmid). Thistranscription vector provides the readily manipulable DNA template fromwhich the RNA polymerase (e.g., T7 RNA polymerase) transcribes asingle-stranded RNA copy of the viral antigenome (or genome) with theprecise, or nearly precise, 5′ and 3′ termini. The orientation of theviral genomic DNA copy and the flanking promoter and ribozyme sequencesdetermines whether antigenome or genome RNA equivalents are transcribed.

Accordingly, in the rescue method a rescue composition is employed. Therescue composition can be varied as desired for a particular need orapplication. An example of a rescue composition is a composition whichcomprises (i) a transcription vector comprising an isolated nucleic acidmolecule which comprises a polynucleotide sequence encoding a genome orantigenome of mumps virus and (ii) at least one expression vector whichcomprises at least one isolated nucleic acid molecule encoding thetrans-acting proteins necessary for encapsidation, transcription andreplication. The transcription and expression vectors are selected suchthat transfection of the rescue composition in a host cell results inthe co-expression of these vectors and the production of the recombinantmumps virus.

As noted above, the isolated nucleic acid molecule comprises a sequencewhich encodes at least one genome or antigenome of a mumps virus. Theisolated nucleic acid molecule may comprise a polynucleotide sequencewhich encodes a genome, antigenome or a modified version thereof. In oneembodiment, the polynucleotide encodes an operably linked promoter, thedesired genome or antigenome, a self-cleaving ribozyme sequence and atranscriptional terminator.

In a preferred embodiment of this invention, the polynucleotide encodesa genome or anti-genome that has been modified from a wild-type mumpsvirus by a nucleotide insertion, rearrangement, deletion orsubstitution. In preferred embodiments, the polynucleotide sequenceencodes a cDNA clone for a recombinant mumps virus. It is submitted thatthe ability to obtain replicating virus from rescue may diminish as thepolynucleotide encoding the native genome and antigenome is increasinglymodified. The genome or antigenome sequence can be derived from that ofany strain of mumps virus. The polynucleotide sequence may also encode achimeric genome formed from recombinantly joining a genome or antigenomeor genes from one or more heterologous sources.

Since the recombinant viruses formed by the methods of this inventioncan be employed as tools in diagnostic research studies or astherapeutic or prophylactic immunogenic compositions, the polynucleotidemay also encode a wild type or an attenuated form of the mumps virusselected. In many embodiments, the polynucleotide encodes an attenuated,infectious form of the mumps virus. In particularly preferredembodiments, the polynucleotide encodes a genome or antigenome of amumps virus having at least one attenuating mutation in the 3′ genomicpromoter region and having at least one attenuating mutation in the RNApolymerase gene, as described by published International patentapplication WO 98/13501, which is hereby incorporated by reference.

In addition to polynucleotide sequences encoding the modified forms ofthe desired mumps genome and antigenome as described above, thepolynucleotide sequence may also encode the desired genome or antigenomealong with one or more heterologous genes or a desired heterologousnucleotide sequence. These variants are prepared by introducing selectednucleotide sequences into a polynucleotide sequence encoding a genome orantigenome of mumps. Preferably, a desired heterologous sequence isinserted within an intergenic region of the mumps genome. However, thedesired heterologous sequence can be inserted within a non-coding regionof the mumps polynucleotide sequence, or inserted between a non-codingregion and a coding region, or inserted at either end of thepolynucleotide sequence. In alternative embodiments a desiredheterologous sequence may be inserted within the coding region of anon-essential gene, or in place of the coding region for a non-essentialgene. The insertion site choice can make use of the 3′ to 5′ gradient ofexpression of mumps virus. The heterologous nucleotide sequence (e.g.gene) can vary as desired. Depending on the application of the desiredrecombinant virus, the heterologous nucleotide sequence may encode aco-factor, cytokine (such as an interleukin), a T-helper epitope, arestriction marker, adjuvant, or a protein of a different microbialpathogen (e.g. virus, bacterium, fungus or parasite), especiallyproteins capable of eliciting a protective immune response. It may bedesirable to select a heterologous sequence that encodes an immunogenicportion of a co-factor, cytokine (such as an interleukin), a T-helperepitope, a restriction marker, adjuvant, or a protein of a differentmicrobial pathogen (e.g. virus, bacterium or fungus) in order tomaximize the likelihood of rescuing the desired mumps virus, orminireplicon virus vector. Other types of non-mumps moieties include,but are not limited to, those from cancer cells or tumor cells,allergens amyloid peptide, protein or other macromolecular components.For example, in certain embodiments, the heterologous genes encodecytokines, such as interleukin-12, which are selected to improve theprophylatic or therapeutic characteristics of the recombinant virus.

Examples of such cancer cells or tumor cells include, but are notlimited to, prostate specific antigen, carcino-embryonic antigen, MUC-1,Her2, CA-125 and MAGE-3.

Examples of such allergens include, but are not limited to, thosedescribed in U.S. Pat. No. 5,830,877 and published International PatentApplication Number WO 99/51259, which are hereby incorporated byreference, and include pollen, insect venoms, animal dander, fungalspores and drugs (such as penicillin). Such components interfere withthe production of IgE antibodies, a known cause of allergic reactions.

Amyloid peptide protein (APP) has been implicated in diseases referredto variously as Alzheimer's disease, amyloidosis or amyloidogenicdisease. The β-amyloid peptide (also referred to as Aβ peptide) is a 42amino acid fragment of APP, which is generated by processing of APP bythe β and γ secretase enzymes, and has the following sequence:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu ValPhe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met ValGly Gly Val Val Ile Ala (SEQ ID NO 97).

In some patients, the amyloid deposit takes the form of an aggregated Aβpeptide. Surprisingly, it has now been found that administration ofisolated Aβ peptide induces an immune response against the Aβ peptidecomponent of an amyloid deposit in a vertebrate host (See PublishedInternational Patent Application WO 99/27944). Such Aβ peptides havealso been linked to unrelated moieties. Thus, the heterologousnucleotides sequences of this invention include the expression of thisAβ peptide, as well as fragments of Aβ peptide and antibodies to Aβpeptide or fragments thereof. One such fragment of Aβ peptide is the 28amino acid peptide having the following sequence (As disclosed in U.S.Pat. No. 4,666,829)

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu ValPhe Phe Ala Glu Asp Val Gly Ser Asn Lys (SEQ ID NO 98).

These heterologous sequences may be used in embodiments of thisinvention that relate to mumps virus vectors, which can be used todeliver varied RNAs, amino acid sequences, polypeptides and proteins toan animal or human. The examples set forth herein demonstrate theability of mumps virus to express one or more heterologous genes (andeven 3, 4, or 5 genes) under control of the mumps virus transcriptionalpromoter. In alternative embodiments, the additional heterologousnucleic acid sequence may be a single sequence of up to 7 to 10 kb,which is expressed as a single extra transcriptional unit. Preferably,the Rule of Six (Calain and Roux, 1993) is followed. In certainpreferred embodiments this sequence may be up to 4 to 6 kb. One may alsoinsert heterologous genetic information in the form of additionalmonocistronic transcriptional units, and polycistronic transcriptionalunits. Use of the additional monocistronic transcriptional units, andpolycistronic transcriptional units should permit the insertion of moregenetic information. In preferred embodiments, the heterologousnucleotide sequence is inserted within the mumps genome sequence as atleast one polycistronic transcriptional unit, which may contain one ormore ribosomal entry sites. In alternatively preferred embodiments, theheterologous nucleotide sequence encodes a polyprotein and a sufficientnumber of proteases that cleaves said polyprotein to generate theindividual polypeptides of the polyprotein.

The heterologous nucleotide sequence can be selected to make use of thenormal route of infection of mumps virus, which enters the body throughthe respiratory tract and can infect a variety of tissues and cells, forexample, salivary glands, lymphoid tissue, mammary glands, the testesand even brain cells. The heterologous gene may also be used to provideagents which are used for gene therapy or for the targeting of specificcells. As an alternative to merely taking advantage of the normal cellsexposed during the normal route of mumps infection, the heterologousgene, or fragment, may encode another protein or amino acid sequencefrom a different pathogen which, when employed as part of therecombinant mumps virus, directs the recombinant mumps virus to cells ortissue which are not in the normal route of mumps virus. In this manner,the recombinant mumps virus becomes a vector for the delivery of a widervariety of foreign genes.

For embodiments employing attenuated mumps viruses, conventional meansare used to introduce attenuating mutations to generate a modifiedvirus, such as chemical mutagenesis during virus growth in cell culturesto which a chemical mutagen has been added, followed by selection ofvirus that has been subjected to passage at suboptimal temperature inorder to select temperature sensitive and/or cold adapted mutations,identification of mutant viruses that produce small plaques in cellculture, and passage through heterologous hosts to select for host rangemutations. An alternative means of introducing attenuating mutationscomprises making predetermined mutations using site-directedmutagenesis. One or more mutations may be introduced. These viruses arethen screened for attenuation of their biological activity in cellculture and/or in an animal model. Attenuated mumps viruses aresubjected to nucleotide sequencing to locate the sites of attenuatingmutations.

A rescued recombinant mumps virus is tested for its desired phenotype(temperature sensitivity, cold adaptation, plaque morphology, andtranscription and replication attenuation), first by in vitro means,such as sequence identification, confirmation of sequence tags, andantibody-based assays.

If the attenuated phenotype of the rescued virus is present, challengeexperiments can be conducted with an appropriate animal model. Non-humanprimates provide the preferred animal model for the pathogenesis ofhuman disease. These primates are first immunized with the attenuated,recombinantly-produced virus, then challenged with the wild-type form ofthe virus.

The choice of expression vector as well as the isolated nucleic acidmolecule which encodes the trans-acting proteins necessary forencapsidation, transcription and replication can vary depending on theselection of the desired virus. The expression vectors are prepared inorder to permit their co-expression with the transcription vector(s) inthe host cell and the production of the recombinant virus under selectedconditions.

A mumps rescue includes an appropriate cell milieu, preferablymammalian, in which T7 RNA polymerase is present to drive transcriptionof the antigenomic (or genomic) single-stranded RNA from the viralgenomic cDNA-containing transcription vector. Eitherco-transcriptionally or shortly thereafter, this viral antigenome (orgenome) RNA transcript is encapsidated into functional templates by thenucleocapsid protein and engaged by the required polymerase componentsproduced concurrently from co-transfected expression plasmids encodingthe required virus-specific trans-acting proteins. These events andprocesses lead to the prerequisite transcription of viral mRNAs, thereplication and amplification of new genomes and, thereby, theproduction of novel viral progeny, i.e., rescue.

In the rescue method of this invention, a T7 RNA polymerase can beprovided by recombinant vaccinia virus. This system, however, requiresthat the rescued virus be separated from the vaccinia virus by physicalor biochemical means or by repeated passaging in cells or tissues thatare not a good host for vaccinia virus. This requirement is avoided byusing as a host cell restricted strain of vaccinia virus (e.g. MVA-T7)which does not proliferate in mammalian cells. Two recombinant MVAsexpressing the bacteriophage T7 RNA polymerase have been reported. TheMVA/T7 recombinant viruses contain one integrated copy of the T7 RNApolymerase under the regulation of either the 7.5K weak early/latepromoter (Sutter et al., 1995) or the 11K strong late promoter (Wyatt etal., 1995).

The host cell, or cell line, that is employed in the transfection of therescue composition can vary widely based on the conditions selected forrescue. The host cells are cultured under conditions that permit theco-expression of the vectors of the rescue composition so as to producethe desired recombinant mumps virus. Such host cells can be selectedfrom a wide variety of cells, including eukaryotic cells, and preferablyvertebrate cells. Avian cells may be used, but preferred host cells arederived from a human cell, such as a human embryonic kidney cell.Exemplary host cells are human 293 cells, A549 cells and Hep2 cells.Vero cells as well as many other types of cells can also be used as hostcells. Other examples of suitable host cells are: (1) Human DiploidPrimary Cell Lines: e.g. WI-38 and MRC5 cells; (2) Monkey Diploid CellLine: e.g. FRhL—Fetal Rhesus Lung cells; (3) Quasi-Primary ContinuousCell Line: e.g. AGMK—African green monkey kidney cells.; (4) otherpotential cell lines, such as, CHO, MDCK (Madin-Darby Canine Kidney),and primary chick embryo fibroblasts (CEF). Some eukaryotic cell linesare more suitable than others for propagating viruses and some celllines do not work at all for some viruses. A cell line is employed thatyields detectable cytopathic effect in order that rescue of viable virusmay be easily detected. In the case of mumps, the transfected cells canbe co-cultured on Vero cells because the virus spreads rapidly on Verocells and makes easily detectable plaques. In general, a host cell whichis permissive for growth of the selected virus is employed.

In alternatively preferred embodiments, a transfection-facilitatingreagent may be added to increase DNA uptake by cells. Many of thesereagents are known in the art. LIPOFECTACE (Life Technologies,Gaithersburg, Md.) and EFFECTENE (Qiagen, Valencia, Calif.) are commonexamples. Lipofectace and Effectene are both cationic lipids. They bothcoat DNA and enhance DNA uptake by cells. Lipofectace forms a liposomethat surrounds the DNA while Effectene coats the DNA but does not form aliposome.

The transcription vector and expression vector can be plasmid vectorsdesigned for expression in the host cell. The expression vector whichcomprises at least one isolated nucleic acid molecule encoding thetrans-acting proteins necessary for encapsidation, transcription andreplication may express these proteins from the same expression vectoror at least two different vectors. These vectors are generally knownfrom the basic rescue methods, and they need not be altered for use inthe improved methods of this invention.

In the method of the present invention, a standard temperature range(about 32° C. to about 37° C.) for rescue can be employed; however, therescue at an elevated temperature has been shown to improve recovery ofthe recombinant RNA virus. The elevated temperature is referred to as aheat shock temperature (See Published International Patent ApplicationNumber WO 99/63064, which is hereby incorporated herein by reference).An effective heat shock temperature is a temperature above the standardtemperature suggested for performing rescue of a recombinant virus atwhich the level of recovery of recombinant virus is improved. Anexemplary list of temperature ranges is as follows: from 38° C. to about47° C., with from about 42° C. to about 46° C. being the more preferred.Alternatively, it is noted that heat shock temperatures of 43° C., 44°C., and 45° C. are particularly preferred.

Numerous means are employed to determine the level of recovery of thedesired recombinant mumps virus. As noted in the examples herein, achloramphenicol acetyl transferase (CAT) reporter gene is used tomonitor and optimize conditions for rescue of the recombinant virus. Thecorresponding activity of the reporter gene establishes the baseline andtest level of expression of the recombinant virus. Other methods includedetecting the number of plaques of recombinant virus obtained andverifying production of the rescued virus by sequencing.

In preferred embodiments, the transfected rescue composition, as presentin the host cell(s), is subjected to a plaque expansion step (i.e.amplification step). The transfected rescue composition is transferredonto at least one layer of plaque expansion cells (PE cells). Therecovery of recombinant virus from the transfected cells is improved byselecting a plaque expansion cell in which the mumps virus or therecombinant mumps virus exhibits enhanced growth. Preferably, thetransfected cells containing the rescue composition are transferred ontoa monolayer of substantially confluent PE cells. The variousmodifications for rescue techniques, including plaque expansion, arealso set forth in Published International Patent Application Number WO99/63064.

The recombinant mumps viruses prepared from the methods of the presentinvention are employed for diagnostic, prophylactic and therapeuticapplications. Preferably, the recombinant viruses prepared from themethods of the present invention are attenuated. The attenuatedrecombinant virus should exhibit a substantial reduction of virulencecompared to the wild-type virus which infects human and animal hosts.The extent of attenuation is such that symptoms of infection will notarise in most individuals, but the virus will retain sufficientreplication competence to be infectious and elicit the desired immuneresponse profile for vaccines. The attenuated recombinant virus can beused alone or in conjunction with pharmaceuticals, antigens, immunizingagents or adjuvants, as vaccines in the prevention or amelioration ofdisease. These active agents can be formulated and delivered byconventional means, i.e. by using a diluent or pharmaceuticallyacceptable carrier.

Accordingly, in further embodiments of this invention attenuatedrecombinantly produced mumps virus is employed in immunogeniccompositions comprising (i) at least one recombinantly producedattenuated mumps virus and (ii) at least one of a pharmaceuticallyacceptable buffer or diluent, adjuvant or carrier. Preferably, thesecompositions have therapeutic and prophylactic applications asimmunogenic compositions in preventing and/or ameliorating mumpsinfection. In such applications, an immunologically effective amount ofat least one attenuated recombinant mumps virus of this invention isemployed in such amount to cause a substantial reduction in the courseof the normal mumps infection.

The formulation of such immunogenic compositions is well known topersons skilled in this field. Immunogenic compositions of the inventionmay comprise additional antigenic components (e.g., polypeptide orfragment thereof or nucleic acid encoding an antigen or fragmentthereof) and, preferably, include a pharmaceutically acceptable carrier.Suitable pharmaceutically acceptable carriers and/or diluents includeany and all conventional solvents, dispersion media, fillers, solidcarriers, aqueous solutions, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The term“pharmaceutically acceptable carrier” refers to a carrier that does notcause an allergic reaction or other untoward effect in patients to whomit is administered. Suitable pharmaceutically acceptable carriersinclude, for example, one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. Pharmaceutically acceptable carriers may furthercomprise minor amounts of auxiliary substances such as wetting oremulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the antigen. The use of such media and agentsfor pharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, use thereof in immunogenic compositions of thepresent invention is contemplated.

Administration of such immunogenic compositions may be by anyconventional effective form, such as intranasally, parenterally, orally,or topically applied to mucosal surface such as intranasal, oral, eye,lung, vaginal, or rectal surface, such as by aerosol spray. Thepreferred means of administration is parenteral or intranasal.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, and thelike.

The vaccine compositions of the invention can include an adjuvant,including, but not limited to aluminum hydroxide; aluminum phosphate;Stimulon™ QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, Mass.);MPL™ (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research,Hamilton, Mont.), IL-12 (Genetics Institute, Cambridge, Mass.);N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine(CGP 19835A, referred to a MTP-PE); and cholera toxin. Others which maybe used are non-toxic derivatives of cholera toxin, including its Bsubunit (for example, wherein glutamic acid at amino acid position 29 isreplaced by another amino acid, preferably, a histidine in accordancewith Published International Patent Application WO 00/18434, which ishereby incorporated herein), and/or conjugates or genetically engineeredfusions of non-mumps polypeptides with cholera toxin or its B subunit,procholeragenoid, fungal polysaccharides.

The recombinantly produced attenuated mumps virus of the presentinvention may be administered as the sole active immunogen in animmunogenic composition. Alternatively, however, the immunogeniccomposition may include other active immunogens, including otherimmunologically active antigens against other pathogenic species. Theother immunologically active antigens may be replicating agents ornon-replicating agents. Replicating agents include, for example,attenuated forms of measles virus, rubella virus, variscella zostervirus (VZV), Parainfluenza virus (PIV), and Respiratory Syncytial virus(RSV).

One of the important aspects of this invention relates to a method ofinducing immune responses in a mammal comprising the step of providingto said mammal an immunogenic composition of this invention. Theimmunogenic composition is a composition which is immunogenic in thetreated animal or human such that the immunologically effective amountof the polypeptide(s) contained in such composition brings about thedesired response against mumps infection. Preferred embodiments relateto a method for the treatment, including amelioration, or prevention ofmumps infection in a human comprising administering to a human animmunologically effective amount of the immunogenic composition. Thedosage amount can vary depending upon specific conditions of theindividual. This amount can be determined in routine trials by meansknown to those skilled in the art.

Certainly, the isolated amino acid sequences for the proteins of themumps virus may be used in forming subunit vaccines. They may also beused as antigens for raising polyclonal or monoclonal antibodies and inimmunoassays for the detection of anti-mumps virus protein-reactiveantibodies. Immunoassays encompassed by the present invention include,but are not limited to those described in U.S. Pat. No. 4,367,110(double monoclonal antibody sandwich assay) and U.S. Pat. No. 4,452,901(western blot), which U.S. patents are incorporated herein by reference.Other assays include immunoprecipitation of labeled ligands andimmunocytochemistry, both in vitro and in vivo.

This invention also provides for a method of diagnosing a mumpsinfection, or identifying a mumps vaccine strain that has beenadministered, comprising the step of determining the presence, in asample, of an amino acid sequence of SEQ ID NOS 2-10. Any conventionaldiagnostic method may be used. These diagnostic methods can easily bebased on the presence of an amino acid sequence or polypeptide.Preferably, such a diagnostic method matches for a polypeptide having atleast 10, and preferably at least 20, amino acids which are common tothe amino acid sequences of this invention.

The nucleic acid sequences disclosed herein can also be used for avariety of diagnostic applications. These nucleic acids sequences can beused to prepare relatively short DNA and RNA sequences that have theability to specifically hybridize to the nucleic acid sequences encodingthe mumps virus proteins. Nucleic acid probes are selected for thedesired length in view of the selected parameters of specificity of thediagnostic assay. The probes can be used in diagnostic assays fordetecting the presence of pathogenic organisms, or in identifying amumps vaccine that has been administered, in a given sample. Withcurrent advanced technologies for recombinant expression, nucleic acidsequences can be inserted into an expression construct for the purposeof screening the corresponding oligopeptides and polypeptides forreactivity with existing antibodies or for the ability to generatediagnostic or therapeutic reagents. Suitable expression controlsequences and host cell/cloning vehicle combinations are well known inthe art, and are described by way of example, in Sambrook et al. (1989).

In preferred embodiments, the nucleic acid sequences employed forhybridization studies or assays include sequences that are complementaryto a nucleotide stretch of at least about 10 to about 20 nucleotides,although at least about 10 to 30, or about 30 to 60 nucleotides can beused. A variety of known hybridization techniques and systems can beemployed for practice of the hybridization aspects of this invention,including diagnostic assays such as those described in Falkow et al.,U.S. Pat. No. 4,358,535.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridizations aswell as in embodiments employing a solid phase. In embodiments involvinga solid phase, the test DNA (or RNA) from suspected clinical samples,such as exudates, body fluids (e.g., amniotic fluid, middle eareffusion, bronchoalveolar lavage fluid) or even tissues, is absorbed orotherwise affixed to a selected matrix or surface. This fixed,single-stranded nucleic acid is then subjected to specific hybridizationwith selected probes under desired conditions. The selected conditionswill depend on the particular circumstances based on the particularcriteria required (depending, for example, on the G+C contents, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, et.). Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

The nucleic acid sequences which encode the mumps virus proteins of theinvention, or their variants, may be useful in conjunction with PCR™technology, as set out, e.g., in U.S. Pat. No. 4,603,102. One mayutilize various portions of any of mumps virus sequences of thisinvention as oligonucleotide probes for the PCR™ amplification of adefined portion of a mumps virus gene, or mumps virus nucleotide, whichsequence may then be detected by hybridization with a hybridizationprobe containing a complementary sequence. In this manner, extremelysmall concentrations of mumps nucleic acid may be detected in a sampleutilizing the nucleotide sequences of this invention.

The following examples are included to illustrate certain embodiments ofthe invention. However, those of skill in the art should, in the lightof the present disclosure, appreciate that many changes can be made inthe specific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

The following examples are provided by way of illustration, and shouldnot be construed as limitative of the invention as describedhereinabove.

EXAMPLES Example 1

Materials and Methods

Cells and viruses. Primary chick embryo fibroblast (CEF) cells wereobtained from SPAFAS Inc., Preston, Conn.), and cultured in Eagle'sBasal Medium (BME) supplemented with 5% fetal calf serum. Hep 2 cells,293 cells, A549, and Vero cells were obtained from the American TypeCulture Collection (ATCC) and grown in Dulbecco's Modified Eagle Medium(DMEM) supplemented with 10% fetal calf serum. The Jeryl Lynn strain ofmumps virus was cultured directly on CEF cells from a vial of Mumpsvax®,Lot Numbers 0089E, 0656J, and 1159H (Merck and Co., Inc., West Point,Pa.). Recombinant vaccinia virus Ankara (MVA-T7), expressingbacteriophage T7 RNA polymerase was obtained from Dr. B. Moss [(NationalInstitutes of Health, Bethesda, Md.), see Wyatt et al., 1995].

1.A. Generation of Mumps Virus Jeryl Lynn Consensus Sequence.

Growth of mumps virus Jeryl Lynn strain stock. Mumps virus Jeryl Lynnstrain was cultured directly from vials of Mumpsvax (lot # 1159H, Merckand Co., Inc.) on primary chick embryo fibroblasts (CEFs, Spafas, Inc.)in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetalcalf serum or in Eagle's Basal Medium (BME) supplemented with 5% fetalcalf serum. CEFs plated on T-75 flasks were infected with resuspendedMumpsvax at an approximate multiplicity of infection (moi) of 0.002 for2 hours at room temperature. The inoculum was removed from the cells andreplaced with fresh media. Cells were incubated at 37° C. for 4 days, atwhich time extensive syncytia and cytopathology was observed. Virus wascollected by scraping the cells into the culture media, followed byfreeze-thawing twice in a dry ice/ethanol bath followed by incubation at37° C. Cell debris was removed by centrifugation at 2,500 rpm in aBeckman GS-6KR centrifuge (Beckman Instruments, Inc., Palo Alto,Calif.). Virus was stored at −80° C.

Isolation of Viral RNA, Amplification, and Sequencing.

Mumps viral RNA was isolated from frozen aliquots of virus using TrizolLS Reagent according to the manufacturer (GibcoBRL, Grand Island, N.Y.).Reverse transcription followed by polymerase chain reaction (RT-PCR) wasperformed using the isolated viral RNA as a template and using the TitanOne-Tube RT-PCR System (Boehringer Mannheim, Indiananpolis, Ind.). Themumps genome was amplified in four separate fragments, using thefollowing primer pairs:

5′-₁ACCAAGGGGAGAATGAATATGGG₂₃ (SEQ ID NO. 95) and

5′-₃₈₇₅CTGAACTGCTCTTACTAATCTGGAC₃₈₅₁ (SEQ ID NO. 82) (3.9 kb product);

5′-₃₇₇₃CTGTGTTACATTCTTATCTGTGACAG₃₇₉₈ (SEQ ID NO. 21) and

5′-₇₇₈₃TGTAACTAGGATCTGATTCCAAGC₇₇₆₀ (SEQ ID NO. 72) (4 kb product);

5′-₇₆₇₈AGAGTTAGATCAGCGTGCTTTGAG₇₇₀₁ (SEQ ID NO. 32) and

5′-₁₁₆₈₅CCTTGGATCTGTTTTCTTCTACCG₁₁₆₆₂ (SEQ ID NO. 62) (4 kb product);

5′-₁₁₅₂₉GTGTTAATCCCATGCTCCGTGGAG₁₁₅₅₂ (SEQ ID NO. 42) and

5′-₁₅₃₈₄ACCAAGGGGAGAAAGTAAAATC₁₅₃₆₃ (SEQ ID NO. 53) (3.9 kb product).The suggested protocol from the manufacturer (Boehringer Mannheim,Indiananpolis, Ind., catalog # 1855476) was followed for the RT and PCRconditions. The PCR products were purified on a 1% agarose gel.

The PCR products were sequenced using an Applied Biosystems (ABI) 377Sequencer (Applied Biosystems, Inc., Foster City, Calif.). Forsequencing purposes, a series of primers was designed which spanned theentire mumps genome as shown in Table 4 below. These primer sequenceswere based on nucleotide sequence information obtained from Genbank fora varying combination of incompletely sequenced mumps virus strains.Using the published sequences, a hypothetical mumps genome sequence wasdevised encoding its proteins and then the primers were generatedtherefrom.

In order to determine properly the sequences at the 5′ and 3′ ends ofthe mumps virus Jeryl Lynn genome, viral genome RNA was ligated at itsends and cDNA was then amplified by PCR across the ligated region. Foreach reaction, 3-5 μg viral RNA was incubated in 10% DMSO, 5× ligationbuffer and deionized water at 83° C. for 3 minutes to denature anysecondary structures, and then placed immediately on ice. T4 RNA ligase(20 Units, New England Biolabs, Inc., Beverly, Mass.) and 40 Units ofRNasin (Promega) were added to give a final ligation mixture of 20 μlwhich was incubated overnight at 16° C. The ligation products werephenol/chloroform-extracted and subjected to RT-PCR using the followingprimer pair which spanned the ligated region of the genome:

5′-₁₅₁₆₆GCGCATTGATATTGACAATGI₁₅₁₈₅ (SEQ ID NO. 52) and

5′-₂₁₆CCCTCCTCACCCCTGTCTTG₁₉₇ (SEQ ID NO. 92) The PCR products weresubjected to a second round of PCR using the following nested primers:

5′-₁₅₂₂₇GAATAAAGACTCTTCTGGC₁₅₂₄₅ (SEQ ID NO. 93)

and 5′-₁₃₈GGTAGTGTCAAAATGCCCCC₁₁₉ (SEQ ID NO. 94). The final PCRproducts were gel-purified and sequenced.

Table 4

Primers for sequencing MUV genome

₁ACCAAGGGGAGAATGAATATGGG₂₃ (SEQ ID NO: 95)

₃₈₅CTCAGCAGGCATGCAAAATC₄₀₄ (SEQ ID NO: 96)

₇₆₅CAAGATACATGCTGCAGCCG₇₈₄ (SEQ ID NO: 13)

₁₁₆₉GTCCTAGATGTCCAAATGCG₁₁₈₈ (SEQ ID NO: 14)

₁₅₄₄GACTTTAGAGCACAGCCTTT₁₅₆₃ (SEQ ID NO: 15)

₁₈₄₁CAATCTAGCCACAGCTAACT₁₈₆₁ (SEQ ID NO: 16)

₂₁₀₇CGTTGCACCAGTACTCATTG₂₁₂₆ (SEQ ID NO: 17)

₂₄₈₄GGCATAGACGGGAATGGAGC₂₅₀₃ (SEQ ID NO: 18)

₃₀₇₂TTCGAGCAACGATTGGCAAAGGC₃₀₉₄ (SEQ ID NO: 19)

₃₇₁₂CCAGCTCCGATAAATATGTC₃₇₃₁ (SEQ ID NO: 20)

₃₇₇₃CTGTGTTACATTCTTATCTGTGACAG₃₇₉₈ (SEQ ID NO: 21)

₄₀₆₂CTGACAGTCAGCATAGGAGA₄₀₈₁ (SEQ ID NO: 22)

₄₃₆₄GAAGTCTGCCTCAATGAGAA₄₃₈₃ (SEQ ID NO: 23)

₄₇₁₆CCAACCCACTGATAACAGCT₄₇₃₅ (SEQ ID NO: 24)

₅₁₈₅CCAGCATTGTCACCGATTAG₅₂₀₄ (SEQ ID NO: 25)

₅₅₄₅CAATACAATGAGGCAGAGAG₅₅₆₄ (SEQ ID NO: 26)

₆₂₂₃TGAATCTCCTAGGGTCGTAACGTC₆₂₄₆ (SEQ ID NO: 27)

₅₉₅₂GAGCAACCATCAGCTCCAAT₅₉₇₁ (SEQ ID NO: 28)

₆₃₃₀CATAACCCTGTATGTCTGGAC₆₃₅₀ (SEQ ID NO: 29)

₆₇₈₃GGATGATCAATGATCAAGGC₆₈₀₂ (SEQ ID NO: 30)

₇₁₇₂GGTAAGACACACTGGTGCTA₇₁₉₁ (SEQ ID NO: 31)

₇₆₇₈AGAGTTAGATCAGCGTGCTTTGAG₇₇₀₁ (SEQ ID NO: 32)

₇₈₈₇GCTGGTGGCCGTATGAACTCC₇₉₀₇ (SEQ ID NO: 33)

₈₃₄₄CAGATTGACCATCACTTGAG₈₃₆₃ (SEQ ID NO: 34)

₈₆₆₀CCTAGTCTCCGGTGGACCCG ₈₆₇₉ (SEQ ID NO: 35)

₉₁₆₆CACTGATATGTTAGAGGGAC₉₁₈₅ (SEQ ID NO: 36)

₉₅₈₃CCGAGAGTCCATGTGTGCTC₉₆₀₂ (SEQ ID NO: 37)

₁₀₀₀₀AGAGGATGACAGATTCGATC₁₀₀₁₉ (SEQ ID NO: 38)

₁₀₄₁₅GAGATAGCAGCCTGCTTTCT₁₀₄₃₄ (SEQ ID NO: 39)

₁₀₈₁₃GCTCAGTCATTCCGAGAAGA₁₀₈₃₂ (SEQ ID NO: 40)

₁₁₁₉₃GTCAGGACATCACTAATGCT₁₁₂₁₂ (SEQ ID NO: 41)

₁₁₅₂₉GTGTTAATCCCATGCTCCGTGGAG₁₁₅₅₂ (SEQ ID NO: 42)

₁₂₀₀₆GCAGTAGTGGTGATGACAAG₁₂₀₂₅ (SEQ ID NO: 43)

₁₂₃₇₅CTCCTATGCATTCTCTAGCT₁₂₃₉₅ (SEQ ID NO: 44)

₁₂₇₉₃GCAGATGGTAAATAGCATCA₁₂₈₁₂ (SEQ ID NO: 45)

₁₃₂₁₉CGATTATGAGATAGTTGTTC₁₃₂₃₈ (SEQ ID NO: 46)

₁₃₆₂₃GTTCATCCGAATCAGCATCC₁₃₆₄₂ (SEQ ID NO: 47)

₁₄₀₃₆CAAGCAGGTATAGCAGCAGG₁₄₀₅₅ (SEQ ID NO: 48)

₄₃₈₈CCGACCCGAATAATCACGAG₁₄₄₀₇ (SEQ ID NO: 49)

₄₇₇₅CATCAGATCATGACACCCTA₁₄₇₉₄ (SEQ ID NO: 50)

₁₄₉₆₃GTGATAACACCCATGGAGATTC₁₄₉₈₄ (SEQ ID NO: 51)

₁₅₁₆₆GCGCATTGATATTGACAATG₁₅₁₈₅ (SEQ ID NO: 52)

₁₅₃₈₄ACCAAGGGGAGAAAGTAAAATC₁₅₃₆₃ (SEQ ID NO: 53)

₁₄₉₇₇CATGGGTGTTATCACGTCTC₁₄₉₅₈ (SEQ ID NO: 54)

₁₄₅₄₉CAACACGCCTCCTCCAGTAC₁₄₅₃₀ (SEQ ID NO: 55)

₁₄₂₀₁GTACACCCTCCAGATCCACA₁₄₁₈₂ (SEQ ID NO: 56)

₁₃₈₀₇CCATGATGTGGATGATAAAC₁₃₇₈₈ (SEQ ID NO: 57)

₁₃₄₁₂CATATTCGACAGTTTGGAGT₁₃₃₉₃ (SEQ ID NO: 58)

₁₃₀₂₁CAAGGTCATATACACATAGT₁₃₀₀₂ (SEQ ID NO: 59)

₁₂₆₀₂CTACACAAGACTCGACAGGT₁₂₅₈₃ (SEQ ID NO: 60)

₁₂₁₉₇CTCCCGCTAATCTGAGTGCT₁₂₁₇₈ (SEQ ID NO: 61)

₁₁₆₈₅CCTTGGATCTGTTTTCTTCTACCG₁₁₆₆₂ (SEQ ID NO: 62)

₁₁₃₈₂CAGATATCTAGACAGCCAGC₁₁₃₆₃ (SEQ ID NO: 63)

₁₁₀₁₇GCACATCTTGCTCACGTTCT₁₀₉₉₈ (SEQ ID NO: 64)

₁₀₆₁₀GGGTAGGATCTGATGGAGGA₁₀₅₉₁ (SEQ ID NO: 65)

₁₀₁₂₂CGACCTGTAGCCTTTATCTC₁₀₁₀₃ (SEQ ID NO: 66)

₉₇₅₃TCATGCCGCATCTCAATGAG₉₇₃₄ (SEQ ID NO: 67)

₉₃₅₆CACCATACTGTAATTGGGCG₉₃₃₇ (SEQ ID NO: 68)

₈₉₆₉ACCCACTCCACTCATTGTTGAACC₈₉₄₆ (SEQ ID NO: 69)

₈₆₀₂TTCAGCTCGAATTGCCTTCC₈₅₈₃ (SEQ ID NO: 70)

₈₄₆₁GAGTATCTCATTTAGGCCCG₈₄₄₂ (SEQ ID NO: 71)

₇₇₈₃TGTAACTAGGATCTGATTCCAAGC₇₇₆₀ (SEQ ID NO: 72)

₇₇₅₆GACAAGAAATGCACTCTGTA₇₇₃₇ (SEQ ID NO: 73)

₇₃₂₅CATCACTGAGATATTGGATC₇₃₀₆ (SEQ ID NO: 74)

₆₉₀₉GATACCGTTACTCCGTGAAT₆₉₈₀ (SEQ ID NO: 75)

₆₃₄₇CAGACATACAGGGTTATGATGAG₆₃₂₅ (SEQ ID NO: 76)

₅₇₅₃GTGACTGCATGATGGTCAGG₅₇₃₄ (SEQ ID NO: 77)

₅₃₅₂CATCTGCATCTCATCTAGCA₅₃₃₃ (SEQ ID NO: 78)

₄₉₅₁CACGTGCATTCGTCTGTGCT₄₉₃₂ (SEQ ID NO: 79)

₄₅₈₉GAAAAGATTGCATAGCCCAAGC₄₅₆₈ (SEQ ID NO: 80)

₄₂₅₆CTGGAGAATAGCACTGGCAG₄₂₃₇ (SEQ ID NO: 81)

₃₈₇₅CTGAACTGCTCTTACTAATCTGGAC₃₈₅₁ (SEQ ID NO: 82)

₃₅₃₀GCACGCTGTCACTACAGGAG₃₅₁₁ (SEQ ID NO: 83)

₃₁₅₈GTGAGTTCATATGGCGCTTC₃₁₃₉ (SEQ ID NO: 84)

₂₇₆₇GCTAGTGTTGTCTTTACTGT₂₇₄₈ (SEQ ID NO: 85)

₂₅₀₇TGAGGCTCCATTCCCGTCTATG₂₄₈₆ (SEQ ID NO: 86)

₂₃₃₄GTTGGTTGGATAGTTGGATC₂₃₁₅ (SEQ ID NO: 87)

₁₇₈₀GCCCACTTGCGACTGTGCGT₁₇₆₁ (SEQ ID NO: 88)

₁₄₃₈CTCATATGCGGCAGCAGGTT₁₄₁₉ (SEQ ID NO: 89)

₁₀₃₉GGATCGGAGCTTAGTGAGTT₁₀₂₀ (SEQ ID NO: 90)

₆₅₆GTACACTGTAACACCGATCC₆₃₇ (SEQ ID NO: 91)

₂₁₆CCCTCCTCACCCCTGTCTTG₉₇ (SEQ ID NO:92)

Prior work had shown that the Jeryl Lynn vaccine strain contained amixture of two distinct virus populations (Afzal et al., 1993).Therefore in order to minimize the potential for sub-optimalprotein-protein interactions (by splicing together cDNA fragmentsderived from the different virus populations into the genome cDNA)during the rescue process, a well isolated plaque from the Jeryl Lynnvaccine preparation (designated as plaque isolate 4, PI 4) was selectedand amplified for the derivation of the full length genome cDNA, and theNP, P and L expression plasmids.

1.B Construction of expression plasmids for MUV NP, P and L proteins.Expression plasmids for the MUV NP, P and L proteins (pMUVNP, pMUVP,pMUVL) were constructed by splicing cDNA for each ORF between the T7 RNApolymerase promoter and the T7 RNA polymerase transcription terminationsequence of a modified plasmid vector pEMC (Moss et al., 1990) whichcontained the cap independent translation enhancer (CITE) ofencephalo-myocarditis virus (EMC). The primers used for RT-PCRamplification of the MUV NP protein ORF, from total MUV infected-cell(CEF) RNA, were 5′ CGTCTC CCATGTTGTCTGTGCTCAAAGC (SEQ ID NO 99) and 5′ATCATTCTCGAG TTGCGATTGGGGTTAGTTTG (SEQ ID NO 100); the resulting cDNAfragment was gel purified, trimmed with BsmBI and XhoI, and then ligatedinto NcoI/XhoI cut pEMC, such that the AUG of the NP protein ORF wasadjacent to the CITE. Primers for the amplification of the MUV P ORFwere 5′ TTCCGGGCAAGCCATGGATC (SEQ ID NO 101) and 5′ ATCATTCTCGAGAGGGAATCATTGTGGCTCTC (SEQ ID NO 102). The P ORF cDNA (modified bysite-directed mutagenesis to include the two G nucleotides which areco-transcriptionally inserted by viral polymerase to generate P mRNA)was also cloned into the NcoI/XhoI sites of pEMC. Because of it's largesize the L protein ORF was assembled in two steps; primers 5′ATCATTCGTCTCCCATGGCGGGCCTAAATGAGATACTC (SEQ ID NO 103) and 5′CTTCGTTCATCTGTTTTGGATCCG (SEQ ID NO 104) were used in the first step to produce acDNA fragment which was trimmed with BsmBI and BamHI, then cloned intothe NcoI/BamHI sites of pEMC. In the second step primers 5′ CAGAGTACCTTATATCGGATCC (SEQ ID NO 105) and 5′ ATCATTCTGCAGGAATTTGGATGTTAGTTCGGCAC (SEQ ID NO 106) were used to amplify a cDNA fragment whichwas cloned into the BamHI/PstI sites of the plasmid from step one above,to complete the L protein ORF. Four cDNA clones for each of the threeORFs were sequenced and the ORF with the highest level of homology tothe Jeryl Lynn consensus nucleotide/amino acid sequence was chosen ineach case for use in rescue experiments.

1.C. Construction of a synthetic MUV minireplicon. Referring to FIG. 1,The T7 RNA polymerase promoter sequence was designed to starttranscription with the exact MUV 5′ terminal nucleotide, and a HDVribozyme sequence (Been et al.) was positioned to generate the preciseMUV 3′ terminal nucleotide in minireplicon RNA transcripts. Duplicate T7RNA polymerase termination signals were included after the HDV ribozymesequence. The bacterial chloramphenicol acetyl transferase (CAT) ORFreplaces all of the coding and intercistronic sequence of the MUVgenome; the remaining essential MUV specific sequence comprises the 3′MUV Leader (55 nt) with adjacent 90 nt NP gene untranslated region(UTR), and the 5′ MUV Trailer (24 nt) adjacent to the 137 nt L gene UTR.

The synthetic MUV minireplicon (MUVCAT) was assembled from cDNArepresenting a modified MUV genome, where all the coding andintercistronic regions were replaced by the CAT ORF. The cDNA for theMUV 3′ and 5′ ends was amplified by RT/PCR from total infected-cell(CEF) RNA, using primer pairs 5′ ACCAAGGGGAGAATGAATATGGG (SEQ ID NO107)/5′ATCATTCGTCTCTTTTCCAGGTAGTGTCAAAATGCC (SEQ ID NO 108), and5′ACCAAGGGGAGAA AGTAAAATC (SEQ ID NO 109)/5′ATCATTCGTCTCTATCGAATAAAGACTCTTCTGGC (SEQ ID NO 110) respectively. In asecond round of PCR amplification nested primers were used for additionof the T7 RNA polymerase promoter and the 5′ to NarI portion of thehepatitis delta virus (HDV) ribozyme sequence to the MUV 5′ and 3′ endsrespectively; these primer pairs were: 5′AAGCTCGGCGGCCGCTTGTAATACGACTCACTATAACCAAGGGGAGAAAGTAAAATC (SEQ ID NO 111)/5′ ATCATTCGTCTCTATCGAATAAAGACTCTTCTGGC (SEQ ID NO 112); for addition of the T7RNA polymerase promoter, and 5′ATCATTGGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCACCAAGG GGAGAATGAATATGGG (SEQ IDNO 113)/5′ ATCATTCGTCTCTTTTCCAGGTAGTGTCAAAATGCC (SEQ ID NO 114) foraddition of the ribozyme component. The CAT ORF cDNA was amplified usingprimers 5′ TCATTCGTCTCGGAAAATGGAGAAAAAAAT CACTGGATATACC (SEQ ID NO 115)and 5′ATCATTCGTCTCTCGATTTA CGCCCCGCCCTGCCACTC (SEQ ID NO 116). All threecomponents were gel purified, trimmed with BsmBI, joined together in afour-way ligation and cloned into the NotI/NarI sites of modified pBSK S(+)(Sidhu et al., 1995) to produce the complete minireplicon plasmid,pMUVCAT.

1.D Construction of a full length genome cDNA for MUV. The full lengthgenome cDNA of MUV (pMUVFL) was assembled 5′ end to 3′ end by thesuccessive cloning of contiguous cDNA fragments into the same plasmidbackbone that was used for the construction of pMUVCAT (See FIG. 2).Each cDNA fragment was amplified from total infected-cell RNA by RT-PCRusing primer pairs which contained suitably unique restriction sites; ineach case the positive sense primer contained a 5′ proximal NotI site inaddition to the virus specific endonuclease site, to facilitate thestep-wise cloning strategy. Prior to addition to the growing full lengthclone, the cDNA fragment spanning the virus 3′ end to the BssHII sitewas assembled separately in pBluescript II SK(+) (Stratagene, La Jolla,Calif.). In the first step the BssHII/ClaI cDNA fragment was cloned intothe ClaI/XhoI sites of pBluescript, using a 5′ extended primer togenerate an XhoI site adjacent to the virus specific BssHII site. In thesecond step the virus 3′ end to ClaI cDNA fragment was cloned into theNotI/ClaI sites of plasmid from the first step to complete the virus 3′end to BssHII sequence. The T7 RNA polymerase promoter sequence wasadded to the virus 3′ end by incorporation into the (+) sense RT/PCRprimer used to generate the virus 3′end/ClaI terminal fragment. The 5′terminal fragment (BamHI/NarI) of the genome cDNA was separatelymodified in a second round of PCR amplification in order to add the5′end to NarI portion of the HDV ribozyme sequence. A total of fourcloning cycles was employed for assembly of pMUVFL; after each round,four clones were sequenced in the region of newly added cDNA andcompared to MUV consensus sequence. The cDNA clone containing the leastnumber of mutations was then selected for addition of the next cDNAfragment. The fully assembled cDNA clone was resequenced to verifystability during bacterial amplification. Electrocompetent SURE cells(Stratagene, La Jolla, Calif.) and DH5alpha cells (GibcoBRL, GrandIsland, N.Y.) were used as bacterial hosts for cloning of MUV cDNA.

1.E Rescue of CAT activity from transfected cells. For rescue of CATactivity, cells were either infected with MUV and transfected with invitro transcribed MUVCAT minireplicon RNA or infected with MVA-T7 andtransfected with pMUVCAT along with pMUVNP, pMUVP and pMUVL expressionplasmids. In vitro transcriptions were carried out with 4 μg of pMUVCATas the template for T7 RNA polymerase in a 20 μl final volume accordingto the manufacturer's protocol (Promega, Madison, Wis.); template DNAwas then digested with RQ-1 DNase. Overnight cultures of 293 cells grownto approximately 80% confluence in six-well dishes were infected withMUV at a moi of 1-2; at 1 hour post infection (hpi) a mixture containing5-10 μl of in vitro transcription reaction (approximately 5-10 μg RNA)and 10-12 μl of LipofectACE (GibcoBRL) was added to each well, accordingto the manufactuer's protocol. At 48 hpi cells were scraped intosuspension, collected by centrifugation, resuspended in 100 μl of 0.25Mtris buffer pH 7.8, and subjected to three rounds of freeze-thaw. Theclarified cell extracts were then assayed for CAT activity using either¹⁴C labelled chloramphenicol (Sidhu et al., 1995) or fluoresceinlabelled chloramphenicol as substrate (Molecular Probes. Eugene, Ore),followed by analysis of reaction products on a Thin Layer Chromatogram.

For rescue of CAT activity in the absence of MUV helper virus, 293, Hep2and A549 cells were grown overnight in six-well dishes to approximately80% confluence, infected with MVA-T7 at an moi of 10 and transfected 1hpi with a mixture containing 200 ng pMUVCAT, 300 ng pMUVNP, 50 ngpMUVP, 200 ng pMUVL, and 10-12 μl of LipofectACE. At 24 hpi thetransfection mixture was replaced with 2 ml of fresh growth medium andcells were incubated for a further 24 hr, followed by preparation ofcell extracts and CAT assay as described above.

1.F Recovery of infectious full length MUV from transfected cells. Forrescue of infectious MUV from cDNA, A549 cells grown overnight toapproximately 90% confluence in six-well dishes were infected withMVA-T7 at an moi of 4; at 1 hpi cells were transfected with a mixturecontaining 3-7ug pMUVFL, 300 ng pMUVNP, 50 ng pMUVP, 200 ng pMUVL and 14μl of Lipofectace. At 24 hpi the transfection mixture was replaced withgrowth medium (DMEM containing 10% fetal calf serum), and cells wereincubated at 37° C. for a further 48 hr; either supernatants (P1) ortotal transfected cell monolayers scraped into suspension were thentransferred directly onto confluent A549 cell monolayers, which wereincubated at 37° C. for four days and then prepared for whole cell ELISA(see below) in order to detect MUV infectious foci. Supernatants (P2)from these A549 indicator cells were further passaged onto confluentVero cell monolayers and incubated at 37° C. for 3-4 days to observe MUVinduced syncytia.

1.G Identification and authentication of rescued MUV. Initialidentification of rescued MUV (rMUV) was carried out using a whole cellELISA; A549 cells infected with transfection supernatants (see above)were fixed with 10% formaldehyde in 1× phosphate buffered saline (PBS)for 30 mins at room temperature; cells were then rinsed once with PBSand once with blocking solution (5% (w/v) milk in x1 PBS), followed byincubation overnight at 4° C. in blocking solution. The overnightblocking solution was then removed and cells were incubated at roomtemperature for 2-3 hr with MUV polyclonal rabbit antiserum (AccessBiomedical, San Diego) diluted 1/400 in fresh blocking solution. Thepolyclonal antiserum was then removed; cells were rinsed 5× withblocking solution and were then incubated at room temperature for 2-3 hrwith horseradish peroxidase (HRP) conjugated goat anti-rabbit serum(DAKO Corporation, Carpinteria, Calif.), diluted 1/1000 in blockingsolution. The goat serum was then removed; cells were washed 5× withblocking solution and 1× with PBS, followed by addition of enough AECsubstrate (DAKO Corporation) to cover cell monolayers, which were thenincubated at 37° C. for 15-20 mins to facilitate stain development.

Nucleotide tags present only in pMUVFL (not present in any laboratorygrown Jeryl Lynn MUV) were verified in rMUV by sequence analysis of cDNAfragments amplified by RT/PCR from Vero cells infected with (P2) rMUV.RNA was prepared from infected cells in a six-well dish by extractionwith Trizol (GibcoBRL) according to the manufacturer's protocol;one-fifth of the total RNA from each well was used as the template forRT/PCR amplification according to directions for the Titan Kit(Boehringer Mannheim, Indianapolis, Ind.), with primer pairs flankingeach of three separate nucleotide tags. The resulting RT/PCR fragmentswere purified from a 1% agarose gel by electroelution, and sequencedusing an Applied Biosystems (ABI) 377 sequencer (Applied Biosystems,Inc., Foster City, Calif.) according to the manufacturer's protocols.

Example 2

Rescue of reporter gene activity from transfected cells. In order tohelp define a system which would permit the rescue of infectious mumpsvirus from cDNA, a mumps virus minireplicon containing the CAT reportergene was assembled. The construct was designed to allow synthesis of aRNA minigenome of negative polarity under control of the T7RNApolymerase promoter. The three terminal G residues of the T7 promoterwere omitted during construction of the minireplicon in order to providea transcriptional start site which began with the precise 5′ nucleotideof the MUV genome. Inclusion of the HDV ribozyme in the minirepliconconstruct permitted cleavage of the T7RNA polymerase transcript toproduce the authentic MUV specific 3′ end. The total number ofnucleotides (966) in the MUVCAT minireplicon RNA was divisible by six,in agreement with the Rule of Six (Calain and Roux, 1993), which statesthat unless the genome length is a multiple of six, efficientreplication will not occur. Expression of the CAT gene was under controlof a MUV specific promoter, and could occur only if minireplicon RNAbecame encapsidated with NP protein and then interacted with functionalMUV specific RNA polymerase proteins.

Recovery of CAT activity was observed here using two different rescuesystems. In the first procedure in vitro transcribed MUVCAT RNA wastransfected into 293 cells which had been previously infected with MUV.Under these conditions rescued CAT activity was usually relatively low,but was reproducible and always well above background levels (See FIG.3A). Panels A1, A2 and A3 show the results from three separate rescueexperiments; panel A1, lane 1 shows CAT activity in MUV-infected cellstransfected without in vitro transcribed pMUVCAT RNA, lane 2 shows CATactivity in MUV-infected cells transfected with RNA transcribed in vitrofrom pMUVCAT; lane 3 shows CAT activity in MUV-infected cellstransfected with RNA transcribed in vitro from pMUVCAT-GG; lane 4 showsCAT activity in uninfected cells transfected with RNA transcribed invitro from pMUVCAT. Each CAT assay shown in panel A1 was carried out at37° C. for 3-4 hrs with 20% of the extract from approximately 10⁶transfected cells. Panel A2 lane 1 shows MUV-infected cells transfectedwith RNA transcribed in vitro from pMUVCAT; lane 2 shows uninfectedcells transfected with RNA transcribed in vitro from pMUVCAT. Each CATassay shown in panel A2 was carried out at 37° C. for 5 hrs using 50% ofthe extract from approximately 10⁶ transfected cells. Panel A3 lane 1shows MUV infected cells transfected with RNA transcribed in vitro frompMUVCAT; lane 2 shows MUV-infected cells transfected without in vitrotranscribed pMUVCAT RNA; lane 3 shows uninfected cells transfected within vitro transcribed RNA from pMUVCAT. Each CAT assay shown in panel A3was carried out at 37° C. for 4 hrs using 50% of the extract fromapproximately 10⁶ transfected cells.

CAT activity could not be rescued from a MUVCAT construct (pMUVCAT-GG)which contained 2 of the 3 additional G residues normally present in theT7RNA polymerase promoter. However, two mutations present in the MUVtrailer region of the same MUVCAT construct prevented conclusiveinterpretation of this observation. Results from these experimentsindicated that nt1-145 and nt15223-15384 of the MUV genome contained thenecessary sequences for genome encapsidation, transcription andpresumably replication. Having defined a minireplicon sequence whichpermitted rescue of CAT activity in the presence of MUV expressed helperproteins, a second system was designed to carry out rescue of CATactivity from transfected DNA, including pMUVCAT, pMUVNP, pMUVP andpMUVL. In this system MUV NP, P and L proteins and MUVCAT minirepliconRNA transcripts were co-expressed inside 293, Hep2, and A549 cells,under control of MVA-T7 induced T7RNA polymerase. Initial experimentscarried out in 293 cells indicated that CAT rescue was efficient andreproducible. Increased efficiency of CAT rescue was seen in Hep2 cellsrelative to 293 cells, and a series of plasmid titrations was performedto optimize the relative amounts of each plasmid in the transfectionmixture. Further increase in rescue efficiency was observed in A549cells relative to Hep2 cells, with almost 100% conversion of substratein a 3 hr CAT assay, using 20% of A549 cell lysate from one well of asix well dish. (FIG. 3B). These results demonstrated that the MUV helperproteins expressed from pMUVNP, pMUVP and pMUVL were sufficient topromote encapsidation, replication and transcription of MUVCAT antisenseRNA minigenomes. Furthermore, the optimal conditions observed for CATrescue provided a starting point for the rescue of infectious MUVentirely from cDNA.

Example 3

Recovery of full length mumps virus from transfected cells. The fulllength MUV cDNA was assembled in such a way as to permit the synthesisof a precise 15,384 nt positive sense RNA copy of the virus genome undercontrol of the T7 RNA polymerase promoter. As with the MUVCATminireplicon, the T7 RNA polymerase promoter sequence was modified toomit the three terminal G residues, providing a transcriptional startsite beginning at the exact MUV terminal nucleotide. The HDV ribozymewas employed to generate the exact MUV 3′ terminal nucleotide of thepositive sense genome transcripts.

To recover MUV from cDNA, A549 cells were infected with MVA-T7 whichexpresses T7 RNA polymerase, and then transfected with pMUVFL, andplasmids expressing the MUV NP, P and L proteins. Results for rescue ofreporter gene activity from the MUVCAT minireplicon along with resultsfrom similar work on the related rubulavirus SV5 (He et al, 1997; Murphyand Parks, 1997) indicated that the MUV NP, P and L proteins would besufficient to encapsidate, replicate and then transcribe the T7 RNApolymerase generated positive sense genome RNA transcripts, provided allthe interacting components were present at operable levels and ratios.A549 cells were chosen for MUV rescue experiments because they supportedMUV replication and more efficient CAT rescue activity than other celllines tested (potentially through more efficient transfection), and theywere also more resistant to MVA-T7 induced cytopathology. In the firstsuccessful rescue experiment, supernatant medium (without clarification)from transfected cells was transferred to fresh A549 indicator cells.Three infectious foci were observed by whole cell ELISA in one out offive wells of indicator cells (FIG. 4). Following passage of supernatantfrom these cells onto a fresh Vero cell monolayer three syncytia wereobserved under the microscope. One of these syncytia was aspirated intomedium as a liquid plaque pick, and used to infect fresh Vero cells;numerous syncytia were then observed on this cell monolayer (FIG. 5),and total infected-cell RNA was extracted for identification of rescuedvirus. In a second rescue experiment at least 10-20 infectious foci wereobtained from each well of transfected cells as seen on indicator cellsstained by whole cell ELISA (FIG. 5). In this experiment all wells,except where pMUVL was omitted from the transfection mixture, containedrescued virus, indicating that the rescue process was very reproducible.The optimal level of each plasmid so far determined for the rescue ofMUV from cDNA is 300 ng pMUVNP, 50 ng pMUVP, 200 ng pMUVL and 3-7 μg ofpMUVFL.

Example 4

Identification of rescued MUV. It was important to demonstrate that rMUVwas derived from pMUVFL. This was made possible by the presence of threenucleotide tags in pMUVFL, introduced by RT/PCR mis-incorporation duringassembly of the full length genome cDNA. These tags differentiatedpMUVFL from both the consensus sequences of the Jeryl Lynn vaccinevirus, and a passaged plaque isolate of the Jeryl Lynn vaccinepreparation from which pMUVFL was derived. Two of the tags representedsilent changes at nucleotides 6081 and 11731 in the F and L genesrespectively; a third tag resulted in a Lys to Arg substitution at aminoacid 22 of the L protein (corresponding to nucleotide position 8502) ofpMUVFL. To show that rMUV was generated from pMUVFL and not from eitherof the other two MUV populations grown in the laboratory, RT/PCRproducts, prepared from rMUV infected-cell RNA, spanning each of thethree nucleotide tags were sequenced at the relevant position(s). Todemonstrate that these RT/PCR products were derived solely from infectedcell RNA, and not from carry-over of trace quantities of transfectingplasmid DNA, one reaction was carried out with rMUV infected cell RNA asthe template for PCR amplification without prior reverse transcription.Results from the RT/PCR amplifications, and subsequent sequencinganalysis of RT/PCR products are shown in FIG. 6. Total RNA was preparedfrom Vero cell monolayers infected with P2 rMUV virus from transfectedcells. RT/PCR reactions were set up to generate cDNA products spanningthe 3 separate nucleotide tag sites present only in pMUVFL and rMUV.Lane 1 shows marker 1 kb ladder (Gibco/BRL); lanes 2,3 and 4 show RT/PCRproducts spanning nucleotide tag positions 6081, 8502 and 11731respectively. To demonstrate these RT/PCR products were not derived fromcontaminating plasmid DNAs, an identical reaction to that used for thegeneration of the cDNA shown in lane 4 was performed without RT; theproduct(s) of this reaction are shown in lane 5. To demonstrate that norMUV could be recovered when pMUVL was omitted from transfectionmixtures, a RT/PCR reaction identical to that used to generate the cDNAproducts shown in lane 4 was set up using Vero cell RNA derived fromtransfections carried out without pMUVL; products from this reaction areshown in lane 6.

The consensus sequence data generated from the RT/PCR products shown inFIG. 6 clearly demonstrate that the rescued MUV contained the samenucleotide tags present only in the full length genome cDNA of MUV (FIG.7). See Table 1 of FIG. 8 for a listing of the nucleotide and amino aciddifferences between the full length cDNA clone and the plaque isolate 4(PI 4) and the consensus sequence for Jeryl Lynn strain (SEQ ID NO 1).

In view of the above examples, it is concluded that infectious mumpsvirus has been produced from a DNA copy of the virus genome. Thisprocedure required the co-transfection of MVA-T7-infected A549 cellswith plasmids encoding MUV NP, P and L proteins, along with a plasmidcontaining the complete genome cDNA of mumps virus. The success of thisprocess was contingent upon the development of a consensus sequence forthe entire mumps virus genome (Jeryl Lynn strain) and the noveldevelopment of a mumps virus minireplicon rescue system.

Note: A Lys to Arg substitution at amino acid 22 of the L protein in thefull length construct did not disrupt obtaining the rescued mumps virus.

Example 5

Mumps Virus as an Expression Vector for One or More Heterologous Genes

The following experiments establish mumps virus as an expression vector.This embodiment is demonstrated by the recovery of infectiousrecombinant mumps virus expressing one or more reporter genes.

Construction of recombinant mumps virus that contain either theBeta-Galactosidase gene, the Firefly Luciferase gene, or the FireflyLuciferase gene and the CAT gene. In order to permit insertion ofheterologous genes or foreign genetic information into the mumps virusgenome, a unique AscI restriction endonuclease site was generated in thefull length genome cDNA, using site directed mutagenesis. The AscI sitewas positioned in the 5′ non-coding region of the M gene (genomenucleotides 4451-4458), such that additional heterologous genescontaining the appropriate flanking regulatory sequences of mumps virusand terminal AscI sites, could be integrated into the mumps genomebetween the virus M and F genes, to produce novel infectious mumps virusrecombinant(s) capable of expressing the foreign gene(s). Mumps virusrecombinants containing either the beta-galactosidase gene or thefirefly luciferase gene have been constructed (see FIG. 11). Anotherrecombinant mumps virus containing the EMC virus CITE adjacent to theluciferase translation initiation codon was also constructed forcomparison with protein (luciferase) levels produced by theluciferase-containing recombinant which utilized the normal mumps viruscis-acting regulatory elements for initiation of translation.

The firefly luciferase gene was prepared for insertion into the mumpsvirus genome by two rounds of nested PCR, using primers whichincorporated mumps virus specific sequences (genome nucleotides4459-4538 and 4392-4449 respectively) adjacent to the ATG and UAA of theluciferase gene. In this process genome nucleotide 4450 was deleted fromthe PCR-generated DNA fragment to maintain the “rule-of-six” in thefinal luciferase-containing recombinant genome; also, in the same DNAfragment, genome nucleotides 4539-4545 were replaced by the sevennucleotides normally found upstream of the luciferase ATG. Terminal AscIsites present in the final PCR product facilitated addition of theluciferase gene and flanking mumps virus specific sequence into themumps virus genome. Similarly, a separate mumps virus recombinantcontaining the beta-galactosidase gene was constructed. ThePCR-generated DNA fragment incorporating the beta-galactosidase gene andflanking mumps virus specific sequences contained the same deletion ofgenome nucleotide 4450, as in the luciferase-containing DNA fragment.However a second TAA trinucleotide was incorporated adjacent to thenormal TAA translation termination codon of the Beta-galactosidase gene,in order to preserve the “rule-of-six” in the final recombinant mumpsvirus genome. Also, unlike the luciferase-containing construct the sevenupstream nucleotides flanking the Beta-galactosidase ATG (genomenucleotides 4539-4545) were mumps virus specific. A third mumps virusrecombinant containing the EMC virus CITE adjacent to the ATG of theluciferase gene, was also constructed. As for the recombinant containingonly the luciferase gene, nested PCR reactions were used to separatelyadd mumps virus specific sequence at the 5′ end and 3′ end of the CITEand luciferase gene, respectively. In a three way ligation, the 3′ endof the CITE and the 5′ end of the luciferase gene were joined at theNcoI restriction endonuclease site and added into the AscI site of themumps virus genome. Genome nucleotide 4450 was deleted, and thetrinucleotide ACT was added to the 5′ end of the CITE during PCR inorder to preserve the “rule-of-six” in the resulting recombinant mumpsvirus.

Mumps virus recombinants were constructed that contained both the CATgene and the luciferase gene, either as two separate transcriptionalunits, or as a single transcriptional unit containing the EMC CITE as aninternal ribosomal entry site for translation of the second gene(luciferase) of the polycistron (see FIG. 12). Nested PCR was used togenerate two DNA fragments, one containing the CAT gene and the otherthe luciferase gene, each flanked with the appropriate mumps virusspecific intergenic cDNA sequence. Both of these fragments were joinedand then ligated into the mumps virus genome cDNA via the AscI sitepreviously used for the insertion of single reporter genes. Similarly,nested PCR was used to separately generate DNA fragments containing theCAT gene and the EMC CITE fused to the luciferase gene, each flankedwith appropriate mumps virus specific intergenic cDNA sequence. Both DNAfragments were joined and ligated into the AscI site of the mumps virusgenome cDNA. The order of reporter genes in both genome constructs was5′ CAT-LUC 3′ and 5′ CAT CITE LUC 3′

Rescue of mumps virus recombinants. Plasmids containing the recombinantmumps virus genomes, along with support plasmids expressing the mumpsvirus NP, P and L proteins were transfected into MVA-T7-infected A549cells, as previously described above in Example 3. Total rescued virusfrom transfected cells was amplified first in fresh A549 cells(Passage1), and subsequently in Vero cells. At Passage 3, rescued viruswas assayed for reporter gene activity.

Assay of reporter gene activity. Reporter gene activity was measuredeither in extracts of cells which had been infected with mumps virusrecombinants or by cytological staining of infected cell monolayers.Extracts from cells infected with mumps virus recombinants containingeither the luciferase gene, or the luciferase gene fused to the EMCvirus CITE were assayed for luciferase activity in a luminomiter(Analytical Luminescence Laboratory, Monolight 2010). The preparation ofcell extracts and luciferase assays were performed according to themanufacturer's protocol for the Enhanced Luciferase Assay Kit(Pharmingen, San Diego, Calif.). Extracts from cells infected with mumpsvirus recombinants containing the beta-galactosidase gene were assayedby cytological staining according to the protocol for the beta-galstaining kit (Promega, Madison, Wis.). Measurement of CAT activity wascarried out on freeze-thaw lysates of infected cells, as previouslydescribed in the above Examples.

Expression of Firefly luciferase by mumps virus. Robust luciferaseactivity was detected in the extracts of cells which had been infectedwith rescued virus. In each case, the rescued virus was derived fromrecombinant mumps virus genomic cDNAs which contained either the fireflyluciferase gene alone or both the CAT gene and the luciferase gene intandem. See FIG. 14, which is a thin layer chromatogram that shows CATactivity present in the extracts of Vero cells which were infected withrMUV containing both the CAT and luciferase genes. Recombinant viruscontaining the CAT and luciferase genes as one transcriptional unit(rMUVC/C/L) were plaque purified (1X) from total rescued virus prior toCAT assay. Rescued recombinant virus containing the CAT and luciferasegenes as individual transcription units (rMUVC/L) was assayed as a totalpopulation without plaque purification. Where indicated in FIG. 14,luciferase activity in Vero cell extracts was also measured for bothrMUVC/C/L and rMUVC/L virus recombinants.

In addition, Table 5 below shows the relative light units (RLU) readoutsfor clonal populations of mumps virus recombinants containing theluciferase gene (rMUV LUC and rMUV CITE-LUC), that were isolated fromrescued virus populations by three successive rounds of plaquepurification. The robust expression of luciferase activity by mumpsvirus recombinants, as shown in Table 5, clearly demonstrates thepotential for mumps virus to express one or more heterologous genes froma recombinant genome(s).

TABLE 5 Quantitation of Luciferase produced by rMUVLUC and rMUVCITE-LUCLUC Total LUC LUC/cell Virus RLU* (pg) (ng) (fg) rMUVLUC-2 2.9 × 10⁵ 8.7pg 300 ng 150 fg rMUVLUC-3 1.3 × 10⁵ 7.9 pg 170 ng  85 fg rMUVLUC-4 2.0× 10⁵ 8.3 pg 400 ng 200 fg rMUVCITE- 0.9 × 10⁵ 6.7 pg 190 ng  95 fgLUC-1 rMUVCITE- 0.2 × 10⁵ 3.2 pg 180 ng  90 fg LUC-2 rMUVCITE- 1.1 × 10⁵7.7 pg 190 ng  95 fg LUC-4 RMUV 0 0  0  0 *Average of two monolayerinfections normalized to 10⁴ input pfu.

Expression of beta-galactosidase by mumps virus. Rescued mumps viruscontaining beta-galactosidase has been identified. Rescued virus wasderived from recombinant mumps virus genomic cDNA containing thebeta-galactosidase gene. Beta-galactosidase activity was evident incells infected by recombinant mumps virus, following direct cytologicalstaining. The intense blue stain of the beta-galactosidase activity waspresent only in cells infected by recombinant mumps virus whichcontained the beta-galactosidase gene. Rescued mumps virus which did notcontain any additional heterologous genes produced clear plaques in thesame staining assay (see FIG. 15). The expression of beta-galactosidaseactivity by recombinant mumps virus further demonstrates the ability ofmumps virus to express relatively large heterologous genes under controlof the mumps virus transcriptional promoter.

Example 6

Determination of the Consensus Sequence for JL5 and JL2

The Jeryl Lynn vaccine strain of mumps virus has been shown to consistof two individual variants, JL5 and JL2 (Afzal et al., 1993). The twovariants, called JL5 and JL2, were shown to exist in a ratio of about 1JL2 to 5 JL5 in the vaccine preparation. Since these variants possesssequence differences in the genome near the SH and HN genes, thisdifference was used to distinguish the variants on the genetic level byisolating pure populations of each and sequencing their entire genomes.

Isolation of JL5 and JL2 Variants from Mumps Virus Jeryl Lynn Strain.

Mumps virus Jeryl Lynn strain was cultured directly on chick embryofibroblasts (CEFs) for one passage. This virus stock was then seriallydiluted in 10-fold increments and used to infect confluent CEFs on6-well plates (Becton Dickinson, Franklin Lakes, N.J.). Cells wereinfected by rocking at room temperature for 1½ hours. The inoculum oneach well was then replaced with an agarose overlay (containing 0.9%agarose [Seaplaque, FMC Bioproducts, Rockland, Me.], minimal essentialmedia [MEM], 0.2 mM non-essential amino acids, 0.2 mg/mlpenicillin/streptomycin, 2% FBS, and 0.3375% sodium bicarbonate). Afterthe overlays solidified at room temperature, the infected cells wereincubated at 37° C. for 6 to 8 days until plaques were visible by eyeand light microscopy.

Individual plaques containing viruses were isolated using sterilePasteur pipettes (VWR Scientific, New York, N.Y.) to remove an agaroseplug over each plaque. The isolated plaques were placed in 1 ml of media(MEM supplemented with 2% FBS, 20 mM HEPES, and 0.1 mg/mlpenicillin/streptomycin), vortexed, and used to infect for a secondround of plaque purification. For subsequent steps, 10, 50, 75, 100, or200 μl of each diluted plaque was used to infect fresh cells on 6-wellplates. Infections, overlays, and plaque isolation were performed asdescribed above. After isolating virus from the second round ofplaquing, the process was repeated a third time.

Viruses isolated from third-round plaques were propagated on CEFs on6-well plates for 4 to 6 days at 37° C. to prepare stocks. Viruses werethen expanded by propagation on CEFs in T-25 flasks. After 5 to 7 days,when the infected cells showed the greatest cytopathology, viruses wereharvested and stored frozen at −80° C.

RT-PCR and Sequencing of Isolated Variants.

RNA isolation and RT-PCR were performed as described in the “Isolationof viral RNA, amplification, and sequencing” section of example 1.A. Thefollowing gene-specific primers were used to amplify portions of the SHand HN genes: ₆₂₂₃TGAATCTCCTAGGGTCGTAACGTC₆₂₄₆ (SEQ ID NO 27) and₈₉₆₉ACCCACTCCACTCATTGTTGAACC₈₉₄₆ (SEQ ID NO 69). Amplified products weregel-purified on 1% agarose and isolated from the gel slices using theWizard PCR Purification Kit (Promega, Madison, Wis.). Amplified productswere then sequenced in the SH gene region [using primers₆₂₂₃TGAATCTCCTAGGGTCGTAACGTC₆₂₄₆ (SEQ ID NO 27,₆₇₈₃GGATGATCAATGATCAAGGC₆₈₀₂ (SEQ ID NO 30),₇₃₂₅CATCACTGAGATATTGGATC₇₃₀₆ (SEQ ID NO 74),₆₉₀₉GATACCGTTACTCCGTGAAT₆₉₈₀ (SEQ ID NO 75)] to identify them as JL5 orJL2.

Preliminary sequence analysis in the SH gene region was performed todefine which purified viruses were JL5 and which were JL2. Initially,all triple-plaque-purified viruses matched JL5. To obtain JL2 isolates,viruses that had been plaque-purified once and stored frozen werescreened by RT-PCR and sequencing in the SH gene region to determinewhether they were JL2 or JL5. Two isolates identified in this manner asJL2-containing plaques were subjected to two additional consecutiverounds of plaque purification. As above, these isolates were expandedtwice in CEFs followed by RNA extraction, amplification, and sequencing.

After defining each plaque isolate as either JL5 or JL2, two separateisolates of each variant were chosen for sequencing the entire genome.RT-PCR was performed on isolated RNA using the following primer pairs toamplify fragments spanning the entire genome: ₁ACCAAGGGGAGAATGAATATGGG₂₃(SEQ ID NO 95) and ₂₅₀₇TGAGGCTCCATTCCCGTCTATG₂₄₈₆ (SEQ ID NO 86),₂₁₀₇CGTTGCACCAGTACTCATTG₂₁₂₆ (SEQ ID NO 17) and₃₈₇₅CTGAACTGCTCTTACTAATCTGGAC₃₈₅₁ (SEQ ID NO 82),₃₇₇₃CTGTGTTACATTCTTATCTGTGACAG₃₇₉₈ (SEQ ID NO 21) and₆₃₄₇CAGACATACAGGGTTATGATGAG₆₃₂₅ (SEQ ID NO 76),₆₂₂₃TGAATCTCCTAGGGTCGTAACGTC₆₂₄₆ (SEQ ID NO 27) and₈₉₆₉ACCCACTCCACTCATTGTTGAACC₈₉₄₆ (SEQ ID NO 69),₇₆₇₈AGAGTTAGATCAGCGTGCTTTGAG₇₇₀₁ (SEQ ID NO 32) and₉₇₅₃TCATGCCGCATCTCAATGAG₉₇₃₄ (SEQ ID NO 67),₉₅₈₃CCGAGAGTCCATGTGTGCTC₉₆₀₂ (SEQ ID NO 37) and₁₁₆₈₅CCTTGGATCTGTTTTCTTCTACCG₁₁₆₆₂ (SEQ ID NO 62),₁₁₅₂₉GTGTTAATCCCATGCTCCGTGGAG₁₁₅₅₂ (SEQ ID NO 42) and₁₃₄₁₂CATATTCGACAGTTTGGAGT₁₃₃₉₃ (SEQ ID NO 58),₁₃₂₁₉CGATTATGAGATAGTTGTTC₁₃₂₃₈ (SEQ ID NO 46) and₁₅₃₈₄ACCAAGGGGAGAAAGTAAAATC₁₅₃₆₃ (SEQ ID NO 53). Amplified products werepurified and sequenced as described in the “Isolation of viral RNA,amplification, and sequencing” section of example 1.A. To determine thesequences of the genomic termini of each virus isolate, the RNA terminiwere ligated, followed by RT-PCR across the junction, and sequencing (asdescribed in Example 1.A).

Sequences were aligned using Sequencher software (Genecodes, Ann Arbor,Mich.). The JL5 and JL2 sequences represent the consensus determined bycomparing both sequenced plaque isolates for each variant. Purified JL5and JL2 viruses were sequenced with the same series of primers as listedin Table 4 of Example 1.A. For both variants, two separate plaqueisolates were sequenced entirely (See SEQ ID NOS 11 and 12 forrespective consensus sequences for JL5 and JL2, plaque 2 for each. Asexpected, a few sequence differences were observed between the two JL5plaque isolates (See table 6) and the two JL2 plaque isolates (See Table7). The consensus sequences of JL5 plaques 1 and 2 differed from JerylLynn consensus sequence by 4 and 3 nucleotides, respectively (See Table6).

The sequence of JL2 contains 413 differences from JL5, spread across theentire genome, as summarized in Table 8. Five of these differences arepresent in the viral 5′ or 3′ leader sequences. A total of 360 sequencedifferences lie within the coding regions of the viral genes; however,only 73 of these differences encode amino acid differences. Theremaining 48 sequence differences lie within the noncoding regions ofthe viral genes. It is of interest to note that there are no sequencedifferences in the intergenic regions or within any of the internalcis-acting signals (i.e. gene start or gene end signals).

TABLE 6 Sequence differences between plaque isolates for JL5. Gene/Jeryl Lynn JL5 JL5 AA Position Consensus Plaque 1 Plaque 2 Amino acidposition 1405 G A A pro (silent) N/420 1685 T C C tyr(T) or his(C) N/5141703 T A T ser(T) or thr(A) N/520 9619 T C C phe (silent) L/394

TABLE 7 Sequence differences between plaque isolates for JL2. Jeryl LynnJL2 JL2 gene/AA Position Consensus Plaque 1 Plaque 2 amino acid position4 A C A NA leader 3352 A C A gln(A) or his M/30 (C) 3508 T T C val(T) orala(C) M/82 3517 T T C val(T) or ala(C) M/85 13467 A G A lys(A) or arg L/1677 (G)

TABLE 8 Summary of sequence differences between JL5 and JL2 variants.Differences between JL5 and JL2 noncoding region Gene 3′ end 5′ endCoding silent Leader 4 — Na na NP 3 9 8 30 P 2 2 14 22 M 2 1 5 17 F 2 612 33 SH 1 6 5 5 HN 4 3 16 35 L 0 7 13 145 Trailer — 1 Na na TOTALS: 18 35  73 287 na = not applicable.

Example 7

Determination of Relative Abundance of JL5 and JL2 in the Jeryl LynnVaccine.

In order to determine the relative ratios of JL5 to JL2 in a vaccine lotof Jeryl Lynn, an assay was developed that exploited sequencedifferences due to a restriction endonuclease polymorphism between thetwo variants. The assay is called mutational analysis by PCR andrestriction endonuclease cleavage (MAPREC). At position 3828(antigenomic sense), there is a BssH II restriction endonucleaserecognition site in the JL5 genome. In JL2, a G to A nucleotidevariation at this site results in a lack of BssH II recognition. RNAfrom a mixed population of JL5 and JL2 was isolated and amplified usingprimers surrounding this site, resulting in a 254 base pair product. Theprimers used were primers ₃₇₀₈CAGGCCAGCGCCGATAAATATG₃₇₂₉ (SEQ ID NO 117)and ₃₉₆₂AATGACACCCTTCTCCATCAGGG₃₉₄₁ (SEQ ID NO 118). The primerscontained identical sequences to both JL5 and JL2; thus, the fragmentsfrom either variant were expected to amplify at equal probability.Furthermore, the first primer listed above contained fluorescein at its5′ end. The fluoresceinated fragment was cleaved with BssH II, andseparated on an acrylamide gel. A FluorImager was used to scan the geland to quantitate the relative abundance of cleaved and uncleavedproducts, which represent JL5 and JL2, respectively. Cleavage with BssHII left a 120-base pair fluorescent product for JL5 and a 254-base pair(i.e. uncleaved) fluorescent product for JL2.

RNA was isolated from ten vaccine vials of mumps virus Jeryl Lynn(Mumpsvax lot # 0656J, Merck and Co., Inc., West Point, Pa.). The RNAwas amplified (by using the above primers) and the PCR products weredigested with BssH II, separated on a gel, and scanned on theFluorImager. The enzyme digestion was performed by adding 5 units ofBssH II (Roche Molecular Biology, Indianapolis, Ind.) to one-fifth ofthe PCR reaction mix and incubating at 50° C. for 2½ hours. The cleavedproducts were then separated on a 6% acrylamide gel that was thenscanned using a FluorImager (Molecular Dynamics, Sunnyvale, Calif.).

Scanned images were quantitated using ImageQuant software (MolecularDynamics, Sunnyvale, Calif.). A series of controls were used asstandards; these samples consisted of pure JL5 and JL2 viruses mixed inthe following ratios based on titers: 99% JL5/1% JL2, 95% JL5/5% JL2,85% JL5/15% JL2, and 75% JL5/25% JL2. RNA was isolated from the mixedviruses and used in the MAPREC procedure. Results from these controlswere used to generate a standard curve for the assay, which was used todetermine the relative percentages of JL5 and JL2 in the vaccinemixtures. In addition, a series of two-fold dilutions of undigested JL5PCR product was used to determine the linear range of the resultsmeasured on the FluorImager. Furthermore, pure JL2 viral RNA was used asa negative control and pure JL5 viral RNA was used as a positivecontrol. The pure JL5 sample also served as a control to determine theefficiency of the BssH II enzyme. The MAPREC assay and quantitation wererepeated three times for reproducibility. The results were averaged overthe three experiments. FIG. 13 shows a representative scanned gel image.The cleaved and uncleaved products are marked with arrows. The uncleavedproduct, which corresponds to JL2, is 254 base pairs long while thecleaved product, which corresponds to JL5, is 120 base pairs in length.To quantitate relative abundance for each scanned gel, values were firstcorrected for background fluorescence and for the amount of undigestedDNA in a pure JL5 control sample. The % JL5 values were determined bydividing the amount of digested DNA by the total of digested andundigested DNA, and by multiplying that value by 100%. For eachexperiment, RNA from a set of mixed JL5 and JL2 viruses was used togenerate a standard curve. The results of the described calculations forthe vaccine samples were plotted on the standard curves to obtain thevalues shown in Table 9. In the final column, the averages for eachvaccine sample are given for the three experiments. An overall averagefor the ten vaccine samples, which was generated by averaging theresults in the last column, is shown at the bottom of the table.

Table 9 summarizes the results for the ten vaccine vials of Mumpsvaxused in this assay. The relative abundance of the two variants withinthe vaccine for these samples was in the range of 73.1% JL5/26.9% JL2 to76.1% JL5/23.9% JL2. The overall average for all ten vaccine samples forall three experiments was 73.9% JL5/26.1% JL2.

TABLE 9 Relative abundance of JL5 and JL2 in Mumpsvax samples. Expt 1Expt 2 Expt 3 Avg. MumpsVax (% JL5/ (% JL5/ (% JL5/ (% JL5/ Sample %JL2) % JL2) % JL2) % JL2) 1 73.7/26.3 72.5/27.5 74.5/25.5 73.6/26.4 274.1/25.9 72.0/28.0 73.3/26.7 73.1/26.9 3 73.0/27.0 76.8/23.2 73.3/26.774.4/25.6 4 73.9/26.1 75.1/24.9 71.2/28.8 73.4/26.6 5 74.6/25.473.9/26.1 70.9/29.1 73.1/26.9 6 76.0/24.0 76.3/23.7 69.8/30.3 74.0/26.07 77.2/22.8 75.9/24.1 70.4/29.6 74.5/25.5 8 76.2/23.8 74.8/25.268.7/31.3 73.2/26.8 9 79.1/20.9 72.1/27.9 77.0/23.0 76.1/23.9 10 78.8/21.2 73.0/27.0 69.7/30.3 73.8/26.2 Overall average: 73.9/26.1

Provided below are a list of references which are incorporated herein.

REFERENCES

-   Afzal, M. A., Pickford, A. R., Forsey, T., Heath, A. B., and    Minor P. D. (1993). The Jeryl Lynn vaccine strain of mumps virus is    a mixture of two distinct isolates. J Gen Virol. 74; 917-920.-   Baron, M. D., and Barrett, T. (1997). Rescue of rinderpest virus    from cloned cDNA. J Virol, 71, 1265-1271.-   Been, M. D., and G. S. Wickham. 1997. Self-cleaving ribozymes of    hepatitis delta virus RNA. Eur. J. Biochem, 247:741-753.-   Boyer, J. C. and Haenni, A. L. (1994). Infectious transcripts and    cDNA clones of RNA viruses. Virology 198, 415-426.-   Buchholz, U. J., Finke, S. and Conzelmann, K. K. (1999). Generation    of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is    not essential for virus replication in tissue culture, and the human    RSV leader region acts as a functional BRSV genome promoter. J.    Virol. 73: 251-259.-   Calain, P. and Roux, L. (1993). The rule of six, a basic feature for    efficient replication of Sendai virus defective interfering RNA. J.    Virol. 67: 4822-4830.-   Collins, P. L., Hill, M. G., Camargo, E., Grosfield, H., Chanock, R.    M., and Murphy, B. R.(1995). Production of infectious human    respiratory syncytial virus from cloned cDNA confirms an essential    role for the transcription elongation factor from the 5″ proximal    open reading frame of the M2 mRNA in gene expression and provides a    capability for vaccine development. Proc. Natl. Acad. Sci. USA 92;    11563-11567.-   Durbin, A. P., Hall, S. L., Siew, J. W., Whitehead, S. S., Collins,    P.-   L. and Murphy, B. R. (1997). Recovery of infectious human    parainfluenza virus type 3 from cDNA. Virology 235, 323-332.-   Elango, N., Varsanyi, T., Kovamees, J., Norrby, E. Molecular cloning    and characterization of six genes, determination of gene order and    intergenic sequences and leader sequence of mumps virus. J. Gen.    Virol. 1988; 69: 2893-2900.-   Fuerst, T. R., Niles, E. G., Studier, F. W. and Moss, B. (1986).    Eukaryotic transient expression system based on recombinant vaccinia    virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl.    Acad. Sci. USA 83: 8122-8126.-   Garcin, D., Pelet, T., Calain, P., Sakai, Y., Shioda, T., Roux, L.,    Curran, J., Kolakofsky, D. (1995). A highly recombinogenic system    for the recovery of infectious Sendia paramyxovirus from cDNA:    generation of a novel copyback nondefective interfering virus.    EMBO J. 14: 6087-6094.-   He, B., Paterson, R. G., Ward, C. D. and Lamb, R. A. (1997).    Recovery of infectious SV5 from cloned DNA and expression of a    foreign gene. Virology, 237: 249-260.-   Hoffman, M. A., and Banerjee, A. K. (1997). An infectious clone of    human parainfluenza virus type 3. J. Virol. 71; 4272-4277.-   Jin, H., D. Clarke, H. Z.-Y. Zhou, X. Cheng, K. Coelingh, M. Bryant,    and S. Li. 1998. Recombinant human respiratory syncytial virus (RSV)    from cDNA and construction of subgroup A and B chimeric RSV.    Virology 251: 206-214.-   Johnson, C. D. and Goodpasture, E. W. (1935). The etiology of mumps.    Am. J. Hyg. 21: 46-57.-   Kyte, J and Doolittle, R. F. (1992). J. Mol. Biol. 157: 105-137.-   Lamb, R. A. and Kolakofsky, D. (1996) Paramyxoviridae. The viruses    and their replication. In “Virology” (B N Fields, D M Knipe, and P M    Howley, Eds.) 3rd edition, Vol1, pp 1177-1204. Raven Press, New    York.-   Lawson, N., Stillman, E., Whitt, M., and Rose, J. (1995).    Recombinant vesicular stomatitis viruses from DNA. Proc. Natl. Acad.    Sci. USA 92; 4471-4481.-   Murphy, S. K. and Parks, G. D. (1997). Genome nucleotide lengths    that are divisible by six are not essential but enhance replication    of defective interfering RNAs of the paramyxovirus simian virus 5.    Virology; 232, 145-157.-   Moss, B., Elroy-Stein, O., Mizukami, T., Alexander, W. A., and    Fuerst, T. R. 1990. New mammalian expression vectors. Nature    348:91-92.-   Paterson, R. G. and Lamb, R. A. RNA editing by G-nucleotide    insertion in mumps virus P-gene mRNA transcripts. J. Virol. 1990;    64: 4137-4145.-   Peeters, B. P. H., deLeeuw, O. S., Koch, G., and    Gielkens A. L. J. (1999) Rescue of Newcastle disease virus from    cloned cDNA: evidence that cleavability of the fusion protein is a    major determinant for virulence. J. Virol. 73: 5001-5009.-   Perrotta, A. T., and Been, M. D. (1991) A pseudoknot-like structure    required for efficient self-cleavage of hepatitis delta virus RNA.    Nature, 350: 434-436.

Racaniello, V. R., and Baltimore, D. (1981). Cloned polioviruscomplementary DNA is infectious in mammalian cells. Science 214:916-918.

-   Radecke, F., Spielhofer, P., Schneider, H, Kaelin, K, Huber, M.,    Dotsch, C., Christiansen, G., and Billeter, M. A. (1995). Rescue of    measles virus from cloned DNA. EMBO J; 14: 5773-5784.-   Sambrook, Fritsch and Maniatis. Molecular Cloning—A Laboratory    Manual. Cold Spring Press (Cold Spring Harbor, N.Y.). 1989.-   Schnell, M. J., Mebatsion, T. and Conzelmann, K. K. (1994).    Infectious rabies viruses from cloned cDNA. EMBO J. 13; 4195-4203.-   Sidhu, M. S., Chan, J., Kaelin, K., Spielhofer, P., Radecke, F.,    Schreider, H., Masurekar, M., Dowling, P. C., Billeter, M. A. and    Udem, S. A. (1995). Rescue of synthetic measles virus minireplicons:    Measles genomic termini direct efficient expression and propagation    of a reporter gene. Virology 208: 800-807.-   Schneider, H., Speilhofer, P., Kaelin, K., Dotsch, C., Radecke, F.,    Sutter, G. and Billeter, M. (1997-February). Rescue of measles virus    using a replication-deficient vaccinia-T7 vector. 64(1): 75-64.-   Sutter, G., and Moss, B., (1992-November). Non-replicating vaccinia    vector efficiently expresses recombinant genes. Proceedings of the    National Academy of Sciences of the United States of America.    89(22): 10847-51.-   Sutter, G., Ohlmann, M. and Erfle, V. Non-replicating vaccinia    vector efficiently expressing bacteriophage T7 RNA    polymerase.(1995-August). FEBS Letters 371 (1): 9-12.-   Takeuchi, K., Tanabayashi, K., Hishiyama, M., Yamada, A. The mumps    virus SH protein is a membrane protein and not essential for virus    growth. Virology 225(1): 156-162, 1996.-   Taniguchi, T., Palmieri, M., and Weissman, C. (1978). QB    DNA-containing hybrid plasmids giving rise to QB phage formation in    the bacterial host. Nature 274, 2293-2298.-   Whelan, S. P., Ball, L. A., Barr, J. N. and Wertz, G. T. (1995).    Efficient recovery of infectious vesicular stomatitis virus entirely    from cDNA clones. Proc. Natl. Acad. Sci. USA 92; 8388-8392.-   Wyatt, L. S., Moss, B. and Rozenblat, S. (1995-June).    Replication-deficient vaccinia virus encoding bacteriophage T7 RNA    polymerase for transient gene expression in mammalian cells.    Virology. 210 (1); 202-5.

1. A method for producing a recombinant mumps virus comprising; in atleast one host cell, conducting transfection or transformation, inmedia, of a rescue composition which comprises (i) a transcriptionvector comprising an isolated nucleic acid molecule which comprises apolynucleotide sequence encoding an antigenome of mumps virus, orvariant polynucleotide sequence thereof, and (ii) at least oneexpression vector which comprises one more isolated nucleic acidmolecule(s) comprising a polynucleotide sequence encoding thetrans-acting proteins (NP, P and L) necessary for encapsidation,transcription and replication; under conditions sufficient to permit theco-expression of said vectors and the production of the recombinantvirus; wherein the isolated nucleic acid molecule encoding an antigenomeof mumps virus comprises the polynucleotide sequence selected from thegroup consisting of SEQ. ID NOS. 1, 11 and 12; and wherein therecombinant virus is harvested.